Abstract Book Cover - Weinstein 2012 - University of Chicago
Abstract Book Cover - Weinstein 2012 - University of Chicago
Abstract Book Cover - Weinstein 2012 - University of Chicago
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<strong>Weinstein</strong><br />
Cardiovascular Development Conference<br />
<strong>Chicago</strong><br />
May 2 nd - 4 th<br />
<strong>2012</strong><br />
<strong>2012</strong> <strong>Weinstein</strong> Cardiovascular<br />
Development Conference<br />
May 2 nd - 4 th , <strong>2012</strong><br />
Sheraton <strong>Chicago</strong> Hotel and Towers<br />
<strong>Chicago</strong>, IL USA
<strong>Weinstein</strong> Cardiovascular Development Conference<br />
<strong>2012</strong><br />
Table <strong>of</strong> Contents<br />
Welcome ............................................................................................................................................................2<br />
General Announcements ............................................................................................................................3<br />
Sheraton Hotel Floor Plans........................................................................................................................4<br />
Conference Schedule...................................................................................................................................7<br />
Keynote Presentation................................................................................................................................ 16<br />
<strong>Weinstein</strong> Cardiovascular Development Conference Charter.................................................. 17<br />
<strong>Weinstein</strong> Conference History............................................................................................................... 18<br />
<strong>2012</strong> <strong>Weinstein</strong> Committee Members ................................................................................................. 19<br />
Sponsors ........................................................................................................................................................ 20<br />
<strong>Abstract</strong>s ........................................................................................................................................................ 21<br />
Oral Sessions ...........................................................................................................................................................21<br />
Moderated Poster Discussions.........................................................................................................................69<br />
General Poster Sessions .....................................................................................................................................79<br />
Author Index................................................................................................................................................171<br />
Registrants ..................................................................................................................................................180<br />
1
Welcome to the <strong>2012</strong> <strong>Chicago</strong> <strong>Weinstein</strong> meeting!<br />
Welcome<br />
The organizing committee would like to welcome you to <strong>Chicago</strong>. This is the 19th <strong>Weinstein</strong> meeting and the<br />
first time it is being held in <strong>Chicago</strong>. <strong>Chicago</strong> is a world-class city with a lot to <strong>of</strong>fer and we hope that everyone<br />
will have time to explore our city. On the river, very close to the Sheraton Hotel, is where the Architectural Boat<br />
Tours embark. This is an excellent way to see the tall buildings in <strong>Chicago</strong>. While most <strong>of</strong> you will be able to<br />
see downtown <strong>Chicago</strong>, the various outlying neighborhoods are also exceptional and we hope you can find time<br />
to visit them. Pilsen, Devon, Wicker Park, Logan Square, Bucktown, South Loop, Old Town, Oak Park and many<br />
others are awaiting you!<br />
This year’s meeting is hosted by members <strong>of</strong> two institutions: The <strong>University</strong> <strong>of</strong> <strong>Chicago</strong> and Northwestern<br />
<strong>University</strong>. Each institution has made significant contribution to the field <strong>of</strong> cardiac development. Since our<br />
institutions are located across the city <strong>of</strong> <strong>Chicago</strong>, the meeting will be held at the centrally located Sheraton<br />
<strong>Chicago</strong> Hotel & Towers located on the <strong>Chicago</strong> River, just east <strong>of</strong> the Magnificent Mile. This is an outstanding<br />
hotel that we hope you will enjoy during your stay.<br />
We have tried to organize the meeting with great care to what we feel are the most timely and relevant areas <strong>of</strong><br />
cardiac development. In addition, following the tradition <strong>of</strong> the <strong>Weinstein</strong> meeting, we organized most <strong>of</strong> the<br />
platform presentations so that trainees would be at the forefront <strong>of</strong> the meeting. New this year, we have added<br />
moderated poster discussions so that attendees can see selected posters and engage in lively, informal<br />
conversations about the topics covered. We want this meeting to be as interactive as possible to promote the<br />
free exchange <strong>of</strong> ideas.<br />
The meeting Banquet will be on Thursday at the House <strong>of</strong> Blues, instead <strong>of</strong> the traditional Saturday night affair<br />
with the hope that more <strong>of</strong> you will be able to attend. The meeting will conclude on Friday night with our keynote<br />
speaker, Cliff Tabin. Persons then staying on will have the weekend to explore <strong>Chicago</strong>.<br />
We hope that you enjoy the 19th <strong>Weinstein</strong> conference,<br />
Sincerely,<br />
The <strong>Weinstein</strong> Organizing Committee,<br />
Eric Svensson, M.D., Ph.D., <strong>University</strong> <strong>of</strong> <strong>Chicago</strong>, Chairman<br />
Robert Dettman, Ph.D., Northwestern <strong>University</strong><br />
Akira Imamoto, Ph.D., <strong>University</strong> <strong>of</strong> <strong>Chicago</strong><br />
Tsutomu Kume, Ph.D., Northwestern <strong>University</strong><br />
Elizabeth McNally, M.D., Ph.D., <strong>University</strong> <strong>of</strong> <strong>Chicago</strong><br />
Ivan Moskowitz, M.D., Ph.D., <strong>University</strong> <strong>of</strong> <strong>Chicago</strong><br />
Marcelo Nobrega, M.D., Ph.D., <strong>University</strong> <strong>of</strong> <strong>Chicago</strong><br />
Hans-Georg Simon, Ph.D., Northwestern <strong>University</strong><br />
2
General Announcements<br />
Oral Presentations<br />
Presentations will be held in the Sheraton Ballroom IV and V on the 4 th level <strong>of</strong> the hotel. Each talk is scheduled<br />
for 15 minutes. This includes 12 minutes for the presentation and 3 minutes for questions. Please stay on time!<br />
Moderated Poster Discussions<br />
Two sessions will be held concurrently in River Exhibition Hall A during each general poster session <strong>of</strong> the<br />
meeting as detailed in the meeting schedule. These sessions will be directed by a moderator, and audience<br />
participation is encouraged!<br />
Posters<br />
All posters will be on display on Wednesday, Thursday, and Friday in River Exhibition Hall A on the first level <strong>of</strong><br />
the hotel. Poster set-up begins on Wednesday at 5:40pm and must be removed by 4pm on Friday. Posters are<br />
numbered according to the abstract book. Authors <strong>of</strong> even numbered abstracts should be present at their<br />
poster on Wednesday evening’s poster session, and authors <strong>of</strong> odd numbered abstracts should be present at<br />
the Thursday afternoon session.<br />
Business Meeting<br />
The Business meeting will be held in the Ohio room on the second level <strong>of</strong> the hotel at 12:30 pm on Thursday.<br />
At this time we will select a site for the 2015 meeting. All are welcome to participate.<br />
Evaluations<br />
Please fill out and return the evaluation forms available at the registration desk. These forms provide important<br />
feedback to the organizers and help the future organizers improve the meeting and address any areas <strong>of</strong><br />
concern.<br />
Drink Tickets and Catering<br />
In your registration materials, you will find<br />
two sets <strong>of</strong> drink tickets. The white<br />
tickets are for purchasing drinks during<br />
the poster session on Wednesday<br />
evening. The red tickets are for<br />
purchasing drinks during the banquet at<br />
the House <strong>of</strong> Blues. Lost tickets will not<br />
be replaced.<br />
Banquet at the House <strong>of</strong> Blues<br />
This year’s meeting banquet will start at<br />
6pm on Thursday evening at the House <strong>of</strong><br />
Blues, 329 North Dearborn Street in<br />
downtown <strong>Chicago</strong>. The House <strong>of</strong> Blues<br />
is an easy 15-minute walk from the hotel<br />
as detailed on the map (“A” marks the<br />
Sheraton Hotel, “B” marks the House <strong>of</strong><br />
Blues).<br />
Acknowledgements<br />
We are grateful to Ms. Tharrie Daniels for<br />
her administrative support <strong>of</strong> this meeting.<br />
3
Sheraton Hotel Floor Plans<br />
Level One – Poster Presentations: River Hall A<br />
Level Two – Business Meeting: Ohio Room<br />
4
Level Three - Lobby<br />
Sheraton Hotel Floor Plans<br />
Level Four – Oral Presentations: Ballroom IV and V<br />
5
Wednesday, May 2 nd<br />
12:00 pm Meeting Check-in<br />
2:00 pm Opening Remarks<br />
Condensed Conference Schedule<br />
2:10-3:40 pm Platform Session 1: Human Genetics/CHD Models<br />
3:40-4:10 pm C<strong>of</strong>fee Break<br />
4:10-5:40 pm Platform Session 2: Cardiomyocyte Growth and Regeneration<br />
5:40-9:00 pm Poster Session 1 and Reception<br />
6:00-7:00 pm Moderated Poster Discussion 1: Cardiac Regeneration<br />
6:00-7:00 pm Moderated Poster Discussion 2: Human Genetics & Disease Models<br />
Thursday, May 3 rd<br />
7:00-8:30 am Continental Breakfast<br />
8:30-10:00 am Platform Session 3: Genomics and Gene Regulation<br />
10:00-10:30 am C<strong>of</strong>fee Break<br />
10:30-12:00 am Platform Session 4 Neural Crest and Vascular Development<br />
12:00-1:30 pm Lunch<br />
12:30-1:30 pm <strong>Weinstein</strong> Business Meeting<br />
1:30-4:00 pm Poster Session 2<br />
2:30-3:30 pm Moderated Poster Discussion 3: Transcriptional Regulation<br />
2:30-3:30 pm Moderated Poster Discussion 4: Coronary Artery Development<br />
4:00-5:30pm Platform Session 5: Coronaries, Epicardium, and Conduction System<br />
6:00-10:00 pm Banquet at the House <strong>of</strong> Blues<br />
Friday, May 4 th<br />
7:00-8:30 am Continental Breakfast<br />
8:30-10:00 am Platform Session 6: Early Heart Formation and Progenitors<br />
10:00-10:30 am C<strong>of</strong>fee Break<br />
10:30-12:00 pm Platform Session 7: Chamber Development<br />
12:00-1:30 pm Lunch<br />
1:00-3:30 pm Poster Session 3<br />
2:15-3:15 pm Moderated Poster Discussion 5: Valve Calcification & Development<br />
2:15-3:15 pm Moderated Poster Discussion 6: Stem Cells<br />
3:30-5:00 pm Platform Session 8: Valve Development<br />
5:00-6:00 pm Keynote Speaker – Dr. Cliff Tabin<br />
6:00-6:15 pm Presentation <strong>of</strong> Next Year’s Venue<br />
Closing Remarks<br />
6
12:00 pm Meeting Check-in<br />
2:00 pm Opening Remarks<br />
Conference Schedule<br />
Wednesday, May 2 nd<br />
2:10-3:40 pm Platform Session 1: Human Genetics/CHD Models<br />
Session Chairs: Beth McNally, <strong>University</strong> <strong>of</strong> <strong>Chicago</strong><br />
Bernice Morrow, Albert Einstein College <strong>of</strong> Medicine<br />
2:10 pm S1.1 Deletion <strong>of</strong> the extracellular matrix protein Adamtsl2 results in a<br />
inter-ventricular septal defect in mice<br />
Dirk Hubmacher, Lauren W.Wang, Suneel S. Apte<br />
Lerner Research Institute, Cleveland Clinic<br />
2:25 pm S1.2 Complex trait analysis <strong>of</strong> ventricular septal defects caused by Nkx2-5 mutation<br />
Claire E. Schulkey, Suk D. Regmi, Patrick Y. Jay<br />
Washington <strong>University</strong> School <strong>of</strong> Medicine<br />
2:40 pm S1.3 Functional analysis <strong>of</strong> novel ZIC3 mutations identified in patients with heterotaxy<br />
Jason Cowan, Muhammad Tariq, Stephanie M. Ware<br />
<strong>University</strong> <strong>of</strong> Cincinnati<br />
2:55 pm S1.4 The Genetic Basis <strong>of</strong> Isolated Tetralogy <strong>of</strong> Fallot<br />
Marcel Grunert, Cornelia Dorn, Markus Schueler, Ilona Dunkel, Jenny Schlesinger,<br />
Siegrun Mebus, Katherina Bellmann, Vladimir Alexi-Meskishvili, Sabine Klaassen,<br />
Katharina Wassilew, Bernd Timmermann, Roland Hetzer, Felix Berger, Silke R.<br />
Sperling<br />
Max Planck Institute for Molecular Genetics<br />
3:10 pm S1.5 An Excess <strong>of</strong> Deleterious Variants in VEGF-A Pathway Genes in Down Syndrome-<br />
Associated Atrioventricular Septal Defects<br />
Christine Ackerman, Adam E. Locke, Eleanor Feingold, Benjamin Reshey, Karina<br />
Espana, Janita Thusberg, Sean Mooney, Lora J. H. Bean, Kenneth J. Dooley, Clifford<br />
Cua, Roger H. Reeves, Stephanie L. Sherman, Cheryl L. Maslen<br />
Oregon Health & Science <strong>University</strong><br />
3:25 pm S1.6 Identification <strong>of</strong> Novel Mutations in a Large Family with Different Forms <strong>of</strong><br />
Congenital Heart Disease<br />
AC Fahed, RC Tanos, IG El-Rassy, FF Bitar, JG Seidman, CE Seidman, GM Nemer<br />
Harvard Medical School and American <strong>University</strong> <strong>of</strong> Beirut, Beirut, Lebanon<br />
3:40-4:10 pm C<strong>of</strong>fee Break<br />
4:10-5:40 pm Platform Session 2: Cardiomyocyte Growth and Regeneration<br />
Session Chairs: Hans-Georg Simon, Northwestern <strong>University</strong><br />
Da-Zhi Wang, Children’s Hospital Boston<br />
4:10 pm S2.1 Cardiac hypertrophy in LEOPARD Syndrome is caused by PTPN11 loss-<strong>of</strong>function<br />
activity in the developing endocardium<br />
Jessica Lauriol, Kimberly Keith, Boding Zhang, Roderick Bronson, Kyu-Ho Lee, and<br />
Maria I. Kontaridis<br />
Beth Israel Deaconess Medical Center<br />
7
4:25 pm S2.2 Epigenetic regulation <strong>of</strong> cardiac hypertrophy<br />
Qing-Jun Zhang, David Maloney, Anton Simeonov, and Zhi-Ping Liu<br />
<strong>University</strong> <strong>of</strong> Texas Southwestern<br />
4:40 pm S2.3 Regulation <strong>of</strong> striated muscle gene expression: A role for Prox1 during cardiac<br />
and skeletal muscle development<br />
Louisa K. Petchey, Paul R. Riley<br />
UCL Institute <strong>of</strong> Child Health, London<br />
4:55 pm S2.4 CIP, a novel cardiac nuclear protein regulates cardiac function and remodeling<br />
Zhan-Peng Huang, Hee Young Seok, Jinghai Chen, and Da-Zhi Wang<br />
Children’s Hospital Boston<br />
5:10 pm S2.5 Yap1 regulates cardiomyocyte proliferation through PIK3CB<br />
Zhiqiang Lin, Alexander von Gisea, Pingzhu Zhou, Jessie Buck, and William Pu<br />
Children’s Hospital Boston<br />
5:25 pm S2.6 A Transitional Extracellular Matrix Instructs Epicardial-mediated Vertebrate Heart<br />
Regeneration<br />
Sarah Mercer, Claudia Guzman, Chia-ho Cheng, Shannon Odelberg, Ken Marx and<br />
Hans-Georg Simon<br />
Northwestern <strong>University</strong> Feinberg School <strong>of</strong> Medicine<br />
5:40-9:00 pm Poster Session 1 and Reception<br />
6:00-7:00 pm Moderated Poster Discussion 1: Cardiac Regeneration<br />
Moderated by Paul Riley<br />
2.7 Cardiomyocyte proliferation contributes to post-natal heart growth in humans<br />
Kevin Bersell, Mariya Mollova, Stuart Walsh, Jainy Savla, Tanmoy DasLala, Shin-<br />
Young Park, Leslie Silberstein, Cris dosRemedios, Dionne Graham, Steven Colan and<br />
Bernhard Kühn<br />
Children's Hospital Boston<br />
2.8 Microarray and Ultrastructure Analyses <strong>of</strong> a Regenerative Myocardium<br />
Pooja Pardhanani, Jeremy Barth, Heather J. Evans-Anderson<br />
Winthrop <strong>University</strong><br />
2.9 Deciphering novel molecular mechanisms that facilitate cardiomyocyte dedifferentiation<br />
and proliferation<br />
Rachel Sarig, Dana Rajchman, Yfat Yahalom, Elad Bassat, Benjamin Geiger, Eldad<br />
Tzahor<br />
Weizmann Institute <strong>of</strong> Science, Israel.<br />
2.10 Tissue specific translational pr<strong>of</strong>iling during zebrafish heart regeneration<br />
Yi Fang, Vikas Gupta, Ravi Karra, Taylor Wahlig, Jennifer Holdway, Jinhu Wang, and<br />
Kenneth D. Poss<br />
Duke <strong>University</strong> Medical Center<br />
6:00-7:00 pm Moderated Poster Discussion 2: Human Genetics & Disease Models<br />
Moderated by Cheryl Maslen<br />
1.7 Novel role <strong>of</strong> altered cardiac function in the progression <strong>of</strong> heart defects in Fetal<br />
Alcohol Syndrome (FAS)<br />
Ganga Karunamuni, Shi Gu, Lindsy Peterson, Zhao Liu, Yong Qiu Doughman, Kersti<br />
Linask, Michael Jenkins, Andrew Rollins, Michiko Watanabe<br />
Case Western Reserve <strong>University</strong><br />
8
1.8 Noonan syndrome associated RAF1 mutant evokes hypertrophic cardiomyopathy<br />
features in human cardiomyocytes in vitro.<br />
Malte Tiburcy, Nicole Dubois, Aleksander Hinek, Seema Mital, Darrell Kotton, Wolfram<br />
Zimmermann, Gordon Keller, Benjamin Neel, Toshiyuki Araki<br />
<strong>University</strong> Health Network and Hospital for Sick Children, Toronto, Canada<br />
1.9 Engineering new mouse models to map dosage-sensitive genes in Down<br />
syndrome congenital heart defects<br />
Eva Lana-Elola, Sheona Watson-Scales, Amy Slender, Louisa Dunlevy, Mike Bennett,<br />
Timothy Mohun, Elizabeth M. C. Fisher, Victor L. J. Tybulewicz<br />
National Institute for Medical Research, London<br />
1.10 Recovery <strong>of</strong> ENU induced mutations causing congenital heart disease using<br />
next-gen sequencing<br />
You Li, Sarosh Fatakia, Ashok Srinivasan, Histao Yagi, Rama Damerla, Bishwanath<br />
Chatterjee, Cecilia W. Lo<br />
<strong>University</strong> <strong>of</strong> Pittsburgh School <strong>of</strong> Medicine<br />
7:00-8:30 am Continental Breakfast<br />
Thursday, May 3 rd<br />
8:30-10:00 am Platform Session 3: Genomics and Regulation<br />
Session Chairs: Marcelo Nobrega, <strong>University</strong> <strong>of</strong> <strong>Chicago</strong><br />
Silke Sperling, Max Planck Institute<br />
8:30 am S3.1 Isolation <strong>of</strong> nuclei from specific cardiac lineages in zebrafish for genome-wide<br />
and epigenomic analyses<br />
Bushra Gorsi and H. Joseph Yost<br />
<strong>University</strong> <strong>of</strong> Utah<br />
8:45 am S3.2 TU-tagging in mice defines dynamic gene expression programs in specific<br />
cardiovascular or other cell types within intact tissues<br />
Leslie Gay, Michael R. Miller, Vidusha Devasthali, P. Brit Ventura, Zer Vue, Heather L.<br />
Thompson, Sally Temple, Hui Zong, Michael D. Cleary, Kryn Stankunas, Chris Q. Doe<br />
<strong>University</strong> <strong>of</strong> Oregon<br />
9:00 am S3.3 Escape <strong>of</strong> Gene Body DNA Methylation in Cardiac Genes is Cooperative with the<br />
Cell Type-specific Expression Patterns<br />
Mayumi Oda, Shinji Makino, Hirokazu Enomoto, Ruri Kaneda, Shinsuke Yuasa and<br />
Keiichi Fukuda<br />
Keio <strong>University</strong>, Tokyo, Japan<br />
9:15 am S3.4 Epigenetic regionalization <strong>of</strong> the developing mouse heart<br />
Scott Smemo, Noboru J. Sakabe, Ivy Aneas, Marcelo A. Nobrega<br />
<strong>University</strong> <strong>of</strong> <strong>Chicago</strong><br />
9:30 am S3.5 The novel mechanism <strong>of</strong> histone-chromatin regulation for cardiomyocytes<br />
regeneration<br />
Ryo Nakamaura, Kazuko Koshiba-Takeuchi, Yuko Tsukahara, Tetsuo Sasano, Mizuyo<br />
Kojima, Tetsushi Furukawa, Hesham A Sadek, Jun K Takeuchi<br />
<strong>University</strong> <strong>of</strong> Tokyo, Tokyo, Japan<br />
9:45 am S3.6 Drosophila microRNA-1 Genetically Interacts with little imaginal discs, a histone<br />
demethylase.<br />
Isabelle N. King<br />
Gladstone Institute <strong>of</strong> Cardiovascular Disease, <strong>University</strong> <strong>of</strong> California, San Francisco<br />
9
10:00-10:30 am C<strong>of</strong>fee Break<br />
10:30-12:00 am Platform Session 4 Neural Crest and Vascular Development<br />
Session Chairs: Tsutomu Kume, Northwestern <strong>University</strong><br />
Frank Conlon, <strong>University</strong> <strong>of</strong> North Carolina<br />
10:30 am S4.1 Early neural crest-restricted Noggin over-expression results in congenital<br />
crani<strong>of</strong>acial and cardiovascular defects<br />
Zachary Neeb, Paige Snider and Simon J. Conway<br />
Indiana <strong>University</strong> School <strong>of</strong> Medicine<br />
10:45 am S4.2 The Role <strong>of</strong> Fibronectin - Integrin Signaling in Pharyngeal Microenvironment in<br />
Modulation <strong>of</strong> Cardiac Neural Crest Cell Development<br />
Dongying Chen and Sophie Astr<strong>of</strong><br />
Thomas Jefferson <strong>University</strong><br />
11:00 am S4.3 Slit3-Robo1/2 signalling controls cardiac innervation and ventricular septum<br />
development by regulating neural crest cell survival<br />
Mathilda T.M. Mommersteeg, William D. Andrews, Alain Chédotal, Vincent M.<br />
Christ<strong>of</strong>fels, John G. Parnavelas<br />
<strong>University</strong> College London, London<br />
11:15 am S4.4 The neural crest contributes to coronary artery smooth muscle formation through<br />
endothelin signaling<br />
Yuichiro Arima, Sachiko Miyagawa-Tomita, Kazuhiro Maeda, Koichi Nishiyama, Rieko<br />
Asai, Ki-Sung Kim, Yasunobu Uchijima, Hisao Ogawa, Yukiko Kurihara and Hiroki<br />
Kurihara<br />
<strong>University</strong> <strong>of</strong> Tokyo, Tokyo, Japan.<br />
11:30 am S4.5 CASTOR directly regulates a novel Egfl7/RhoA pathway to promote blood vessel<br />
development and morphogenesis<br />
Marta Szmacinski, Kathleen Christine, Nirav Amin, Joan Taylor, Frank L. Conlon<br />
<strong>University</strong> <strong>of</strong> North Carolina at Chapel Hill<br />
11:45 am S4.6 Nephronectin regulates axial vein morphogenesis in zebrafish<br />
Chinmoy Patra, Filomena Ricciardi, Iva Nikolic, Mirko HH Schmidt, Benno Jungblut,<br />
Felix B. Engel<br />
Max Planck Institute, Germany<br />
12:00-1:30 pm Lunch<br />
12:30-1:30 pm <strong>Weinstein</strong> Business Meeting<br />
1:30-4:00 pm Poster Session 2<br />
2:30-3:30 pm Moderated Poster Discussion 3: Transcriptional Regulation<br />
Moderated by Sylvia Evans<br />
3.7 Mutual Regulation <strong>of</strong> Nkx2.5 and Fibulin-1 in the SHF Regulatory Network<br />
Anthony J. Horton, Marion A. Cooley, Waleed O. Twall, Victor M. Fresco, Boding<br />
Zhang, Christopher D. Clark, W. Scott Argraves and Kyu-Ho Lee<br />
Medical <strong>University</strong> <strong>of</strong> South Carolina<br />
3.8 FOG-2 Mediated Recruitment <strong>of</strong> the NuRD Complex Regulates Cardiomyocyte<br />
Proliferation during Heart Development<br />
Audrey Jerde, Zhiguang Gao, Spencer Martens, Judy U. Early, Eric C. Svensson<br />
The <strong>University</strong> <strong>of</strong> <strong>Chicago</strong><br />
10
3.9 A strict lineage boundary between the first and second heart fields is defined by<br />
the contribution <strong>of</strong> the Tbx5 lineage<br />
Joshua D. Wythe, W. Patrick Devine, Kazuko Koshiba-Takeuchi, Kyonori Togi, Benoit<br />
G. Bruneau<br />
Gladstone Institute <strong>of</strong> Cardiovascular Disease<br />
3.10 Tbx5-Hedgehog pathway is required in second heart field cardiac progenitors for<br />
atrial septation<br />
Linglin Xie, Andrew D. H<strong>of</strong>fmann, Ozanna Burnicka-Turek, Joshua M. Friedland-Little,<br />
Ke Zhang, Ivan P. Moskowitz<br />
The <strong>University</strong> <strong>of</strong> <strong>Chicago</strong><br />
2:30-3:30 pm Moderated Poster Discussion 4: Coronary Artery Development<br />
Moderated by David Bader<br />
5.7 The BMP regulator BMPER is necessary for normal coronary artery formation<br />
Laura Dyer and Cam Patterson<br />
<strong>University</strong> <strong>of</strong> North Carolina at Chapel Hill<br />
5.8 Epicardial chemokine signaling influences ventricular wall proliferation and<br />
coronary vasculogenesis<br />
Susana Cavallero, Hua Shen, Peng Li, Henry M. Sucov.<br />
Keck School <strong>of</strong> Medicine, <strong>University</strong> <strong>of</strong> Southern California<br />
5.9 The role <strong>of</strong> Pod1/Tcf21 in epicardium-derived cells during cardiac development<br />
and fibrosis<br />
Caitlin M. Braitsch, Michelle D. Combs, Susan E. Quaggin, and Katherine E. Yutzey<br />
Cincinnati Children’s Hospital Medical Center<br />
5.10 Epicardial GATA factors regulate coronary endothelial migration via Sonic<br />
hedgehog signaling<br />
Kurt D. Kolander, Mary L. Holtz, and Ravi P. Misra<br />
Medical College <strong>of</strong> Wisconsin<br />
4:00-5:30pm Platform Session 5: Coronaries, Epicardium, and Conduction<br />
System<br />
Session Chairs: Ivan Moskowitz, <strong>University</strong> <strong>of</strong> <strong>Chicago</strong><br />
William Pu, Children’s Hospital Boston<br />
4:00 pm S5.1 Ets-1 Regulates the Migration <strong>of</strong> Coronary Vascular Precursors and Coronary<br />
Endothelial Cell Proliferation<br />
Gene H. Kim, Saoirse S. McSharry, Zhiguang Gao, Ankit Bhatia, Judy Earley, and Eric<br />
C. Svensson<br />
The Universtiy <strong>of</strong> <strong>Chicago</strong><br />
4:15 pm S5.2 Wnt signaling in the developing murine epicardium and epicardium-derived-cells<br />
(EPDCs)<br />
Carsten Rudat, Julia Norden, Makoto M. Taketo, Andreas Kispert<br />
Hannover Medical School, Germany<br />
4:30 pm S5.3 Determination <strong>of</strong> Cardiac Pacemaker Cell Origins Reveals a Critical Role for Wnt<br />
Signaling During their Cell Fate Specification.<br />
Michael Bressan, Gary Liu, and Takashi Mikawa<br />
<strong>University</strong> <strong>of</strong> California San Francisco<br />
11
4:45 pm S5.4 A Tbx5-Scn5a Molecular Network Modulates Function <strong>of</strong> the Cardiac Conduction<br />
System<br />
David E Arnolds, Fang Liu, Scott Smemo, John P Fahrenbach, Gene H Kim, Ozanna<br />
Burnicka-Turek, Elizabeth M McNally, Marcelo M Nobrega, Vickas V Patel, and Ivan P<br />
Moskowitz<br />
The <strong>University</strong> <strong>of</strong> <strong>Chicago</strong><br />
5:00 pm S5.5 Mouse cardiac T-box targets reveal an Scn5a/10a enhancer functionally affected<br />
by genetic variation in humans<br />
Phil Barnett, Malou van den Boogaard, L. Y. Elaine Wong, Federico Tessadori, Martijn<br />
L. Bakker, Connie R. Bezzina, Peter A.C. ‘t Hoen, Jeroen Bakkers, Vincent M.<br />
Christ<strong>of</strong>fels<br />
<strong>University</strong> <strong>of</strong> Amsterdam<br />
5:15 pm S5.6 Activation <strong>of</strong> voltage-dependent anion channel 2 suppresses Ca 2+ -induced<br />
cardiac arrhythmia<br />
Hirohito Shimizu, Johann Schredelseker, Jie Huang, Kui Lu, Sarah Franklin, Kevin<br />
Wang, Thomas Vondriska, Joshua Goldhaber, Ohyun Kwon, Jau-Nian Chen<br />
<strong>University</strong> <strong>of</strong> California, Los Angeles<br />
6:00-10:00 pm Banquet at the House <strong>of</strong> Blues<br />
7:00-8:30 am Continental Breakfast<br />
Friday, May 4 th<br />
8:30-10:00 am Platform Session 6: Early Heart Formation and Progenitors<br />
Session Chairs: Eric Svensson, <strong>University</strong> <strong>of</strong> <strong>Chicago</strong><br />
Robert Kelly, Aix-Marseille <strong>University</strong><br />
8:30 am S6.1 Quantitative analysis <strong>of</strong> polarity in 3D reveals local cell coordination in the<br />
embryonic mouse heart<br />
Jean-François Le Garrec, Chiara Ragni, Sorin Pop, Alexandre Dufour, Jean-Christophe<br />
Olivo-Marin, Margaret Buckingham and Sigolène Meilhac<br />
Institut Pasteur, Paris, France<br />
8:45 am S6.2 Clonally dominant cardiomyocytes direct heart morphogenesis<br />
Vikas Gupta and Kenneth D. Poss<br />
Duke <strong>University</strong> Medical Center<br />
9:00 am S6.3 Gα 13 is required for S1pr2-mediated myocardial migration by regulating<br />
endoderm morphogenesis<br />
Ding Ye, Songhai Chen, Fang Lin<br />
<strong>University</strong> <strong>of</strong> Iowa<br />
9:15 am S6.4 A Cdc42 associated genetic network directs heart lumen formation and<br />
morphogenesis in Drosophila<br />
Georg Vogler, Jiandong Liu and Rolf Bodmer<br />
Sanford-Burnham Medical Research Institute, La Jolla, CA<br />
9:30 am S6.5 Identification and characterization <strong>of</strong> a multipotent cardiac precursor<br />
W. Patrick Devine, Josh D. Wythe, Benoit G. Bruneau<br />
Gladstone Institute <strong>of</strong> Cardiovascular Disease<br />
12
9:45 am S6.6 Distinct origin and commitment <strong>of</strong> HCN4+ First Heart Field cells towards<br />
cardiomyogenic cell lineages<br />
Daniela Später, Monika Abramczuk, Jon Clarke, Kristina Buac, Makoto Sahara,<br />
Andreas Ludwig, Kenneth R. Chien<br />
Massachusetts General Hospital<br />
10:00-10:30 am C<strong>of</strong>fee Break<br />
10:30-12:00 pm Platform Session 7: Chamber Development<br />
Session Chairs: Akira Imamoto, <strong>University</strong> <strong>of</strong> <strong>Chicago</strong><br />
Deborah Yelon, <strong>University</strong> <strong>of</strong> California, San Diego<br />
10:30 am S7.1 Irx1a Acts Downstream <strong>of</strong> nkx Genes in Maintaining Cardiac Chamber Identity in<br />
Zebrafish<br />
Sophie Colombo, Vanessa George, Thomas Schell, Lilianna Solnica-Krezel, Deborah<br />
Yelon, and Kimara L. Targ<strong>of</strong>f<br />
College <strong>of</strong> Physicians & Surgeons, Columbia <strong>University</strong><br />
10:45 am S7.2 Zebrafish second heart field development relies on early specification <strong>of</strong><br />
progenitors and nkx2.5 function<br />
Burcu Guner-Ataman, Noelle Paffett-Lugassy, Meghan S. Adams, Kathleen R. Nevis,<br />
Caroline E. Burns, C. Ge<strong>of</strong>frey Burns<br />
Massachusetts General Hospital<br />
11:00 am S7.3 Notch1 mediated signaling cascades regulate cardiomyocyte polarity and<br />
ventricular wall formation.<br />
Wenjun Zhang, Hanying Chen, Momoko Yoshimoto, Jin Zhang, Mervin C Yoder, Nadia<br />
Carlesso, Weinian Shou<br />
Indiana <strong>University</strong> School <strong>of</strong> Medicine<br />
11:15 am S7.4 Mutations in the NOTCH pathway regulator MIB1 cause ventricular noncompaction<br />
cardiomyopathy<br />
Guillermo Luxán, Jesús C. Casanova, Beatriz Martínez-Poveda, Donal MacGrogan,<br />
Álvaro Gonzalez-Rajal, Belén Prados, Gaetano D'Amato, David Dobarro, Carlos<br />
Torroja, Fernando Martinez, José Luis Izquierdo-García, Leticia Fernández-Friera,<br />
María Sabater-Molina, Young-Y. Kong, Gonzalo Pizarro, Borja Ibañez, Constancio<br />
Medrano, Pablo García-Pavía, Juan R. Gimeno, Lorenzo Montserrat, Luis J. Jiménez-<br />
Borreguero & José Luis de la Pompa<br />
Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain<br />
11:30 am S7.5 The early role <strong>of</strong> Tbx1 in anterior and posterior second heart field cells<br />
M. Sameer Rana, Karim Mesbah, Jorge N. Dominguez, Niels H<strong>of</strong>schreuder, Virginia E.<br />
Papaioannou, Antoon F. Moorman, Vincent M. Christ<strong>of</strong>fels, Robert G. Kelly<br />
Aix-Marseille <strong>University</strong>, Marseille, France<br />
11:45 am S7.6 Retinol Dehydrogenase 10: Roles in Embryo Pharyngeal Patterning and a Model<br />
to Understand How Retinoid Gradients Prevent Congenital Birth Defects<br />
Muriel Rhinn, Pascal Dollé, Richard Finnell, and Karen Niederreither<br />
The <strong>University</strong> <strong>of</strong> Texas, Austin<br />
12:00-1:30 pm Lunch<br />
13
1:00-3:30 pm Poster Session 3<br />
2:15-3:15 pm Moderated Poster Discussion 5: Valve Calcification & Development<br />
Moderated by Andy Wessels<br />
8.7 Analysis <strong>of</strong> TGFβ3 function in valve remodeling and aortic valve calcification<br />
Alejandra C Encinas, Robert Hinton, and Mohamad Azhar<br />
Indiana <strong>University</strong> School <strong>of</strong> Medicine<br />
8.8 Nuclear exclusion <strong>of</strong> Sox9 in valve interstitial cells is associated with calcific<br />
phenotypes in heart valves<br />
Danielle J. Huk, Harriet Hammond, Robert. B. Hinton, and Joy Lincoln<br />
The Research Institute at Nationwide Children’s Hospital, Columbus<br />
8.9 Runx2 is<strong>of</strong>orms have divergent roles in development and pathology in the heart<br />
Andre L.P. Tavares, Jesse A. Brown, Katarina Dvorak and Raymond B. Runyan<br />
<strong>University</strong> <strong>of</strong> Arizona<br />
8.10 3D cardiac valve tissue reconstruction and quantitative evaluations <strong>of</strong> tissue &<br />
cell phenotypes in the Pdlim7 knock-out mouse.<br />
Rudyard W. Sadleir, Robert W. Dettman, Jennifer Krcmery, Brandon Holtrup, and<br />
Hans-Georg Simon<br />
Northwestern <strong>University</strong> Feinberg School <strong>of</strong> Medicine<br />
2:15-3:15 pm Moderated Poster Discussion 6: Stem Cells<br />
Moderated by Richard Harvey<br />
6.7 Dynamic Mesp1 regulation directs cardiac myocyte formation in embryonic stem<br />
cells<br />
Yu Liu, Xueping Xu, Austin Cooney, Robert Schwartz<br />
<strong>University</strong> <strong>of</strong> Houston<br />
6.8 Human amniocytes contain subpopulations <strong>of</strong> stem cells that have a repressed<br />
cardiogenic status<br />
Colin T. Maguire, Bradley Demarest, Jennifer Akiona, Jonathon Hill, James Palmer,<br />
Arthur R. Brothman, H. Joseph Yost, Maureen L. Condic<br />
<strong>University</strong> <strong>of</strong> Utah Molecular Medicine Program<br />
6.9 The Homeobox Transcription Factor, Irx4, Identifies a Mutipotent, Ventricularspecific<br />
Cardiac Progenitor<br />
Daryl Nelson, Joshua Trzebiatowski, Erin Willing, Pearl Hsu, Kevin Schoen, Kevin<br />
Liberko, Matthew Butzler, Pat Powers, Manorama John, Timothy Kamp, Gary Lyons,<br />
<strong>University</strong> <strong>of</strong> Wisconsin-Madison<br />
6.10 Elastin regulates smooth muscle cell differentiation in human inducedpluripotent<br />
stem cells in Williams-Beuren syndrome<br />
Caroline Kinnear, Wing Y. Chang, Karen Kennedy, Aleksander Hinek, Tadeo<br />
Thompson, Shahryar Khattak, Sylvie Gervier, Maryam Niapour, Naila Mahmut, Yanting<br />
Wang, Gordon Keller, William L. Stanford, James Ellis, Seema Mital<br />
Hospital for Sick Children, Toronto, Canada<br />
14
3:30-5:00 pm Platform Session 8: Valve Development<br />
Session Chairs: Robert Dettman, Northwestern <strong>University</strong><br />
Joy Lincoln, Nationwide Children’s Hospital<br />
3:30 pm S8.1 Computational modeling <strong>of</strong> endocardial cell activation during AV valve formation<br />
A.K. Lagendijk, A. Szabo, R. Merks, and J. Bakkers<br />
Hubrecht Institute and <strong>University</strong> Medical Center, Utrecht, The Netherlands<br />
3:45 pm S8.2 Tie1 is Required for Semilunar Valve Form and Function<br />
Chris Brown, Xianghu Qu, Kate Violette, M-K Sewell, W. David Merryman, Bin Zhou,<br />
and H. Scott Baldwin<br />
Vanderbilt <strong>University</strong><br />
4:00 pm S8.3 In Vivo Reduction Of Smad2 Concomitant With Proteoglycan Accumulation<br />
Results In High Penetrance Of Bicuspid Aortic Valves<br />
Loren Dupuis, Robert B. Hinton and Christine B. Kern<br />
Medical <strong>University</strong> <strong>of</strong> South Carolina<br />
4:15 pm S8.4 Filamin-A Regulates Tissue Remodeling <strong>of</strong> Developing Cardiac Valves via a Novel<br />
Serotonin Pathway<br />
Kimberly Sauls, Annemarieke de Vlaming, Katherine Williams, Andy Wessels, Robert<br />
A. Levine, Susan A Slaugenhaupt, Roger R. Markwald, Russell A. Norris<br />
Medical <strong>University</strong> <strong>of</strong> South Carolina<br />
4:30 pm S8.5 Epicardially-derived Fibroblasts Preferentially Contribute to the Parietal Leaflets<br />
<strong>of</strong> the Atrioventricular Valves in the Murine Heart<br />
Andy Wessels, Maurice J. B. van den H<strong>of</strong>f, Richard F. Adamo, Aimee L. Phelps, Marie<br />
M. Lockhart, Kimberly Sauls, Laura E. Briggs, Russell A. Norris, Bram van Wijk, Jose<br />
M. Perez-Pomares, Robert W. Dettman, and John B. E. Burch<br />
Medical <strong>University</strong> <strong>of</strong> South Carolina<br />
4:45 pm S8.6 Endothelial nitric oxide signaling regulates Notch1 in aortic valve development<br />
and disease<br />
Kevin Bosse, Chetan P. Hans, Ning Zhao, Sara N. Koenig, Nianyuan Huang, Anuradha<br />
Guggilam, Ge Tao, Pamela A. Lucchesi, Joy Lincoln, Brenda Lilly and Vidu Garg<br />
Nationwide Children’s Hospital, Columbus<br />
5:00-6:00 pm Keynote Speaker – Dr. Cliff Tabin<br />
Harvard Medical School<br />
6:00-6:15 pm Presentation <strong>of</strong> Next Year’s Venue<br />
Closing Remarks<br />
END OF MEETING<br />
15
Keynote Presentation<br />
Clifford J. Tabin, PhD<br />
George Jacob and Jacqueline Hazel Leder Pr<strong>of</strong>essor<br />
Chairman <strong>of</strong> the Department <strong>of</strong> Genetics<br />
Harvard Medical School<br />
Boston, Massachusetts<br />
Clifford J. Tabin, PhD, pr<strong>of</strong>essor and chairman <strong>of</strong> the Department <strong>of</strong> Genetics<br />
at Harvard Medical School, is considered one <strong>of</strong> the world leaders in current<br />
developmental biology. His pioneering work on Sonic hedgehog signaling (1)<br />
and the molecular pathway that controls the left and right sides <strong>of</strong> the body<br />
(2,3), revealed basic mechanistic insights into the emergence <strong>of</strong> anatomical<br />
form and organization during vertebrate development. These findings have<br />
contributed critically to our understanding <strong>of</strong> congenital limb malformations<br />
and asymmetry-related defects, including those <strong>of</strong> the heart.<br />
Following the footsteps <strong>of</strong> his father, Cliff Tabin attended the <strong>University</strong> <strong>of</strong> <strong>Chicago</strong> as an undergraduate and<br />
expected to launch a career in physics. However, by the time he began his PhD studies at the Massachusetts<br />
Institute <strong>of</strong> Technology (MIT) in Cambridge, the recombinant DNA molecular biology revolution was beginning.<br />
“It was clear that this new technology was going to allow us to do things that no one had ever done before and<br />
understand things in a totally new way,” Cliff Tabin recalled. He turned his focus, joined the laboratory <strong>of</strong> Dr.<br />
Robert Weinberg, a pioneer in the study <strong>of</strong> retroviruses and oncogenes to do his doctoral thesis. Cliff Tabin was<br />
the first researcher at MIT to construct a retrovirus vector, and during his studies he uncovered the genetic<br />
changes in oncogenic RAS, inserted mutant RAS into his retrovirus vector, and demonstrated that it could cause<br />
cancer in mice (4).<br />
Over the past two decades the Tabin lab has contributed significantly to our understanding <strong>of</strong> the development<br />
<strong>of</strong> a wide variety <strong>of</strong> tissues in the embryo, including the forming skeleton. However, he did not stop there, he is<br />
also a leader in uncovering how changes in the regulation <strong>of</strong> genes during embryonic growth is altered through<br />
the course <strong>of</strong> animal evolution to produce the variety <strong>of</strong> forms seen in the natural world. For example, he sent<br />
students to collect data from finches in the Galapagos and in translating the findings to the chicken embryo, they<br />
identified that the expression <strong>of</strong> Bmp4 and calmodulin determines differences in beak size (5,6). Another<br />
expedition led students to central Mexico to collect blind fish from caves, as “Caves provide a unique<br />
environment for evolution,” he said. He even sent students as far as the Gobi Desert to collect organisms for his<br />
evolutionary studies. However, Cliff Tabin has never been to those places himself. “I enjoy the photographs and<br />
live vicariously,” he said.<br />
“I have never been to Nepal, either,” Cliff Tabin added. Nevertheless, he followed through on an philanthropic<br />
opportunity his brother Ge<strong>of</strong>f got him into, and became chairman <strong>of</strong> an international advisory board to the Patan<br />
<strong>University</strong> <strong>of</strong> Health Sciences, a new medical school that he and a group <strong>of</strong> Nepalese physicians established in<br />
Kathmandu, to train physicians to serve in rural districts. Cliff Tabin and his team developed the curriculum for<br />
the medical school, and he has been actively recruiting volunteers from Harvard to teach courses until the<br />
Nepalese faculty develops the expertise to take them over.<br />
Cliff Tabin’s honors and awards number many and include the <strong>2012</strong> Conklin Medal <strong>of</strong> the Society for<br />
Developmental Biology and the 2008 March <strong>of</strong> Dimes Prize in Developmental Biology. Furthermore, he became<br />
an elected member <strong>of</strong> the National Academy <strong>of</strong> Sciences in 2007 and <strong>of</strong> the American Academy <strong>of</strong> Arts and<br />
Sciences in 2000, and he was the recipient <strong>of</strong> the National Academy <strong>of</strong> Sciences Award in Molecular Biology in<br />
1999.<br />
16
<strong>Weinstein</strong> Cardiovascular Development Conference Charter<br />
Scope <strong>of</strong> the Conference<br />
The <strong>Weinstein</strong> Cardiovascular Development Conference is an annual meeting for scientists investigating normal<br />
and abnormal development <strong>of</strong> the heart and vasculature as it may ultimately relate to human disease. It is a<br />
freestanding meeting, unaffiliated with any society or parent organization. Interested individuals or groups from<br />
host institutions organize it on a rotating basis. The intent <strong>of</strong> the meeting is to advance the overall field <strong>of</strong><br />
cardiovascular development through the sharing <strong>of</strong> information and the facilitation <strong>of</strong> collaborative investigations.<br />
True to the vision <strong>of</strong> Dr. Constance <strong>Weinstein</strong>, who first organized this conference, the meeting is intended to<br />
include as many perspectives as possible. Investigators in any relevant area that can provide contributions to<br />
our understanding <strong>of</strong> heart and vascular development are welcome to contribute.<br />
Organization <strong>of</strong> the Conference<br />
In order to provide continuity and to maintain quality the conference, the participants <strong>of</strong> the 1998 meeting voted<br />
to form an organizing committee called the "<strong>Weinstein</strong> Committee". The makeup <strong>of</strong> the committee is composed<br />
<strong>of</strong> a single representative from each <strong>of</strong> the three previous local organizing committees, a single member from<br />
the current host site local organizing committee, and a single representative from each <strong>of</strong> the next two proposed<br />
meeting sites. In addition, two "At-Large" members, who are selected by a vote by the conference participants,<br />
will serve a three-year term. The charge to the Committee is to assist the local organizing committee with<br />
meeting arrangements and organization and to help secure funding. Any institution should have a maximum <strong>of</strong><br />
one member serving on the <strong>Weinstein</strong> Committee at any given time.<br />
In addition, the Committee is charged with soliciting and vetting nominations for future meeting sites and host<br />
institutions. Prospective host institutions should bid to host a future <strong>Weinstein</strong> meeting three years prior to the<br />
year that they desire to host the meeting. Prospective host institutions should submit a preliminary application to<br />
the two At-Large <strong>Weinstein</strong> Committee members at least one month prior to bidding to host a future meeting.<br />
The preliminary application should contain details <strong>of</strong> the prospective local organizing committee, prospective site<br />
for the meeting, and a fundraising plan. The purpose <strong>of</strong> early submission <strong>of</strong> a preliminary application is to allow<br />
the At-Large members <strong>of</strong> the committee to resolve any potential issues or missing details prior to review <strong>of</strong> the<br />
applications by the entire <strong>Weinstein</strong> Committee at the annual business meeting. The <strong>Weinstein</strong> Committee will<br />
evaluate all bids for feasibility to host the conference effectively in terms <strong>of</strong> fundraising and organizational and<br />
scientific capacity. The <strong>Weinstein</strong> Committee will select a maximum <strong>of</strong> three bids to be put to a vote the<br />
following day by all conference attendees. Meeting sites will be selected by vote such that the future local<br />
organizing committee will have a three-year lead-time. The <strong>Weinstein</strong> Committee may solicit additional<br />
applications from prospective host institutions as needed. In the event that multi-year funding is sought from the<br />
National Institutes <strong>of</strong> Health or other national sources, the <strong>Weinstein</strong> Committee will participate in this process.<br />
Local Organizing Committee<br />
To provide a varied flavor and the opportunity for new approaches, each host institution will form a local<br />
organizing committee to select a meeting venue and format and to participate in fundraising. The site should be<br />
selected for its potential to optimize informal communication and interaction. As a way to emphasize new and<br />
topical information, organizers from the host institution should select speakers from among the submitted<br />
abstracts. Scheduling should include opportunities for new voices and encourage the development <strong>of</strong> students,<br />
fellows, and younger faculty. Ample time for discussion is to be provided.<br />
Obligations <strong>of</strong> the Participants<br />
One <strong>of</strong> the most important aspects <strong>of</strong> the <strong>Weinstein</strong> Conference has been the willingness <strong>of</strong> the participants to<br />
share new and unpublished information. This has provided opportunities for the participants to devise new<br />
experiments and develop new hypotheses in a collaborative manner. It is expected that all participants will<br />
participate in a collegial and ethical manner with respect to information obtained at the <strong>Weinstein</strong> Conference.<br />
Permission should be obtained before disclosure <strong>of</strong> another investigator's unpublished data.<br />
Similarly, investigators pursuing similar experiments should inform a presenter if the divulged information has a<br />
bearing on their own work. All participants in the conference should be willing to share their expertise and<br />
reagents in the collective advancement <strong>of</strong> the area <strong>of</strong> cardiovascular development.<br />
17
Annual Business Meeting<br />
Each <strong>Weinstein</strong> Conference will include time set aside for a business meeting and time for a subsequent vote<br />
on a future conference site by conference participants. At the Business Meeting, <strong>Weinstein</strong> Committee members<br />
may consider changes in the direction <strong>of</strong> the conference or its organization. At the 1999 meeting in Tucson,<br />
Arizona, this Charter was distributed to the participants and ratified. Its provisions commenced at the business<br />
meeting <strong>of</strong> the 1999 Tucson, Arizona Conference. The Charter will remain in effect until modified by a vote <strong>of</strong><br />
the <strong>Weinstein</strong> Committee at the annual business meeting.<br />
<strong>Weinstein</strong> Conference History<br />
The <strong>Weinstein</strong> Cardiovascular Development Conference is a continuation <strong>of</strong> an annual meeting <strong>of</strong> investigators<br />
funded by three separate RFAs on Cardiac Development in 1986, 1988, and 1990 at the National Heart, Lung<br />
and Blood Institute <strong>of</strong> the National Institutes <strong>of</strong> Health. Grants funded under the RFA mechanism resulted in a<br />
total <strong>of</strong> 26 research programs in the area <strong>of</strong> Cardiac Development. A separate program <strong>of</strong> Specialized Centers<br />
in Research (SCORs) in Congenital Heart Disease resulted in the addition <strong>of</strong> investigators from the Universities<br />
<strong>of</strong> Iowa, Pennsylvania and Rochester. Investigators were brought together annually at the National Institutes <strong>of</strong><br />
Health to discuss their latest research results and discuss potential areas <strong>of</strong> collaboration. The first meeting was<br />
held in a basement meeting room at the NIH and included approximately a dozen participants. These annual<br />
meetings at the NIH between 1986 and 1993 continued to grow as new research programs were funded and by<br />
word <strong>of</strong> mouth with collaborators. These meetings coincided with the emergence <strong>of</strong> molecular biological and<br />
genetic approaches to the investigation <strong>of</strong> heart development. Upon the expiration <strong>of</strong> the last <strong>of</strong> the RFA<br />
programs, there was a strong interest in keeping these collegial and informative meetings going. Independent<br />
meetings have been held every year since 1994.<br />
Dr. Constance <strong>Weinstein</strong> <strong>of</strong> the NHLBI was instrumental in focusing the attention <strong>of</strong> the NIH on the need for<br />
research in this area. She obtained the funds for the RFA and SCOR programs and organized the intramural<br />
meetings held at the NIH. To honor Dr. <strong>Weinstein</strong>, in 1995, the meeting was formally named the <strong>Weinstein</strong><br />
Cardiovascular Development Conference. Dr. <strong>Weinstein</strong> is retired from NHLBI but she attends these<br />
conferences to monitor progress in the field that she spent so much effort fostering.<br />
<strong>Weinstein</strong> Conference Host Institutions and Sites<br />
Medical <strong>University</strong> <strong>of</strong> South Carolina (Charleston, 1994)<br />
<strong>University</strong> <strong>of</strong> Rochester (Rochester, 1995)<br />
<strong>University</strong> <strong>of</strong> Pennsylvania (Philadelphia, 1996)<br />
<strong>University</strong> <strong>of</strong> Cincinnati (Cincinnati, 1997)<br />
Vanderbilt <strong>University</strong> (Nashville, 1998)<br />
<strong>University</strong> <strong>of</strong> Arizona (Tucson, 1999)<br />
Washington <strong>University</strong> (St. Louis, 2000)<br />
<strong>University</strong> <strong>of</strong> Texas, Southwestern Medical School (Dallas, 2001)<br />
<strong>University</strong> <strong>of</strong> Utah (Salt Lake City, 2002)<br />
Harvard <strong>University</strong> (Cambridge, 2003)<br />
Leiden <strong>University</strong> (Leiden, Netherlands, 2004)<br />
<strong>University</strong> <strong>of</strong> Arizona (Tucson, 2005)<br />
<strong>University</strong> <strong>of</strong> South Florida (Tampa/St. Petersburg, 2006)<br />
Indiana <strong>University</strong> School <strong>of</strong> Medicine (Indianapolis, 2007)<br />
Texas A&M <strong>University</strong> (Houston, 2008)<br />
<strong>University</strong> <strong>of</strong> California, San Francisco (San Francisco, 2009)<br />
<strong>University</strong> <strong>of</strong> Amsterdam, Netherlands (Amsterdam, 2010)<br />
<strong>University</strong> <strong>of</strong> Cincinnati (Cincinnati, 2011)<br />
<strong>University</strong> <strong>of</strong> <strong>Chicago</strong> and Northwestern <strong>University</strong> (<strong>Chicago</strong>, <strong>2012</strong>)<br />
<strong>University</strong> <strong>of</strong> Arizona (Tucson, 2013)<br />
Centro Nacional de Investigaciones Cardiovasculares (Madrid, 2014)<br />
18
National <strong>Weinstein</strong> Committee<br />
<strong>2012</strong> <strong>Weinstein</strong> Committee Members<br />
(Term expiration indicated in parentheses)<br />
Brian L. Black, Ph.D., <strong>University</strong> <strong>of</strong> California, San Francisco (<strong>2012</strong>)<br />
Organizer, 2009 <strong>Weinstein</strong> Conference<br />
Maurice van den H<strong>of</strong>f, Ph.D, Heart Failure Research Center, Amsterdam (2013)<br />
Organizer, 2010 <strong>Weinstein</strong> Conference<br />
Katherine Yutzey, Ph.D., <strong>University</strong> <strong>of</strong> Cincinnati (2014)<br />
Organizer, 2011 <strong>Weinstein</strong> Conference<br />
Eric Svensson, M.D., Ph.D., <strong>University</strong> <strong>of</strong> <strong>Chicago</strong> (2015)<br />
Organizer, <strong>2012</strong> <strong>Weinstein</strong> Conference<br />
Brad Davidson, Ph.D., <strong>University</strong> <strong>of</strong> Arizona (2016)<br />
Organizer, 2013 <strong>Weinstein</strong> Conference<br />
Jose Luis De la Pompa, Ph.D., Centro Nacional de Investigaciones Madrid (2017)<br />
Organizer, 2014 <strong>Weinstein</strong> Conference<br />
Paul Riley, Ph.D., UCL- Institute <strong>of</strong> Child Health (2014)<br />
Member at Large<br />
Deborah Yelon, Ph.D., <strong>University</strong> <strong>of</strong> California, San Diego (2014)<br />
Member at Large<br />
Local Organizing Committee Members<br />
Eric Svensson, M.D., Ph.D., <strong>University</strong> <strong>of</strong> <strong>Chicago</strong>, Chairman<br />
Robert Dettman, Ph.D., Northwestern <strong>University</strong><br />
Akira Imamoto, Ph.D., <strong>University</strong> <strong>of</strong> <strong>Chicago</strong><br />
Tsutomu Kume, Ph.D., Northwestern <strong>University</strong><br />
Elizabeth McNally, M.D., Ph.D., <strong>University</strong> <strong>of</strong> <strong>Chicago</strong><br />
Ivan Moskowitz, M.D., Ph.D., <strong>University</strong> <strong>of</strong> <strong>Chicago</strong><br />
Marcelo Nobrega, M.D., Ph.D., <strong>University</strong> <strong>of</strong> <strong>Chicago</strong><br />
Hans-Georg Simon, Ph.D., Northwestern <strong>University</strong><br />
Tharrie Daniels, Administrative Assistant<br />
19
Sponsors<br />
The <strong>2012</strong> <strong>Weinstein</strong> Cardiovascular Development Conference is hosted by the <strong>University</strong> <strong>of</strong> <strong>Chicago</strong> and<br />
Northwestern <strong>University</strong>. Continuing <strong>Weinstein</strong> meeting support is also provided by a grant form the National<br />
Heart, Lung, and Blood Institute and the National Institute <strong>of</strong> Child Health and Human Development.<br />
Additional financial support was provided by the following:<br />
Gold Sponsors (support <strong>of</strong> > $5,000):<br />
The Company <strong>of</strong> Biologists<br />
March <strong>of</strong> Dimes Foundation<br />
Visualsonics<br />
Silver Sponsors (support <strong>of</strong> $1,000 to $4,999)<br />
ABCAM<br />
American Association <strong>of</strong> Anatomists<br />
American Heart Association<br />
Intavis<br />
Northwestern <strong>University</strong><br />
Bronze Sponsors (support <strong>of</strong> < $1,000)<br />
Integrated DNA Technologies<br />
Genesis: Journal <strong>of</strong> Genetics and Development<br />
Leica Microsystems<br />
Media Cybernetics<br />
Mouse Specifics<br />
National Swine Research and Resource Center<br />
Numira Biosciences<br />
W. Nuhsbaum<br />
20
<strong>Abstract</strong>s<br />
Oral Sessions<br />
Platform Session 1: Human Genetics/CHD Models<br />
S1.1 Deletion <strong>of</strong> the extracellular matrix protein Adamtsl2 results in a inter-ventricular septal defect in<br />
mice<br />
Dirk Hubmacher, Lauren W.Wang, Suneel S. Apte<br />
Department <strong>of</strong> Biomedical Engineering, Lerner Research Institute, Cleveland Clinic<br />
Geleophysic dysplasia (GD) is a rare human genetic disorder presenting with short stature, joint contractures<br />
and thick skin. High morbidity, and frequently, juvenile mortality results from cardiac valvular and tracheopulmonary<br />
anomalies. Mutations in the extracellular proteins ADAMTSL2 or fibrillin-1 lead to recessive or<br />
dominant GD, respectively. Excess TGFβ signaling was described in cells derived from GD patients and may<br />
constitute a major pathogenetic mechanism. ADAMTSL2 directly interacts with latent transforming growth factorβ<br />
binding protein (LTBP)-1 and fibrillin-1. Fibrillin-1 and -2 assemble into micr<strong>of</strong>ibrils and modulate extracellular<br />
TGFβ and BMP signaling. The Adamtsl2 gene was deleted in mice by an intragenic IRES-lacZ-neomycin<br />
cassette, which also provided an expression reporter. Adamtsl2 deletion resulted in neonatal lethality due to an<br />
inter-ventricular septal defect (VSD) and lung abnormalities. β -gal reporter staining showed Adamtsl2<br />
expression within the embryonic heart and lung, consistent with the observed anomalies. Cardiac expression<br />
was primarily localized to the crest <strong>of</strong> the inter-ventricular septum, the atrio-ventricular valve annulus and<br />
coronary vessels. Histology revealed a VSD in 80% <strong>of</strong> the mice with enhanced, localized fibrillin-1 and -2<br />
staining at the crests <strong>of</strong> the inter-ventricular septum. In vitro, recombinant ADAMTSL2 bound to both fibrillin-1<br />
and -2, and accelerated the biogenesis <strong>of</strong> fibrillin-1 micr<strong>of</strong>ibril formation in fetal bovine nuchal ligament cells,<br />
while blocking fibrillin-2 assembly. This suggests that ADAMTSL2 may provide a mechanism to actively regulate<br />
the ratio <strong>of</strong> fibrillin-1 to fibrillin-2 in micr<strong>of</strong>ibrils, with potential consequences for TGFβ/BMP signaling. These<br />
studies suggest a new role for extracellular matrix in regulation <strong>of</strong> cardiac development.<br />
21
S1.2 Complex trait analysis <strong>of</strong> ventricular septal defects caused by Nkx2-5 mutation<br />
Claire E. Schulkey, Suk D. Regmi, Patrick Y. Jay<br />
Departments <strong>of</strong> Pediatrics and Genetics, Washington <strong>University</strong> School <strong>of</strong> Medicine, St. Louis, MO.<br />
Congenital heart disease is a complex trait. The same deleterious mutation typically causes widely varying<br />
presentations because additional, poorly defined factors modify phenotype. Historically, the modifiers have<br />
received less scrutiny than the causes. Nevertheless, the factors, especially ones that reduce risk, may suggest<br />
preventive strategies that a focus on monogenic causes has not. Hence, we assessed the role <strong>of</strong> genetic and<br />
environmental factors on the incidence <strong>of</strong> ventricular septal defects (VSD) caused by an Nkx2-5 heterozygous<br />
knockout mutation. We phenotyped >3100 hearts from a second generation intercross <strong>of</strong> the inbred mouse<br />
strains C57BL/6 and FVB/N. Genetic linkage analysis mapped loci with LOD scores <strong>of</strong> 5-7 on chromosomes 6, 8<br />
and 10 that influence the susceptibility to membranous VSD in Nkx2-5 +/- animals. The chromosome 6 locus<br />
overlaps one for muscular VSD susceptibility. Multiple logistic regression analysis for environmental variables<br />
revealed that maternal age is correlated with the risk <strong>of</strong> membranous and muscular VSD in Nkx2-5 +/- but not wildtype<br />
animals. The maternal age effect is unrelated to aneuploidy or a genetic polymorphism in the progeny.<br />
Ovarian transplant experiments between young and old females indicate that the basis <strong>of</strong> the maternal age effect<br />
resides in the mother and not the oocyte. Experiments to characterize a potential metabolic basis <strong>of</strong> the effect<br />
strongly suggest that the VSDs caused by Nkx2-5 mutation can be prevented. Enumerable factors contribute to<br />
the presentation <strong>of</strong> a congenital heart defect. Their characterization in a mouse model <strong>of</strong>fers the opportunity to<br />
define unanticipated pathways and to develop strategies for prevention.<br />
22
S1.3 Functional analysis <strong>of</strong> novel ZIC3 mutations identified in patients with heterotaxy<br />
Jason Cowan, Muhammad Tariq, Stephanie M. Ware<br />
<strong>University</strong> <strong>of</strong> Cincinnati<br />
Loss <strong>of</strong> function mutations in the zinc finger in cerebellum 3 (ZIC3) transcription factor result in heterotaxy, a<br />
condition characterized by abnormal left-right positioning <strong>of</strong> thoraco-abdominal organs and a wide variety <strong>of</strong><br />
congenital anomalies, particularly <strong>of</strong> the cardiovascular system. Mutations in ZIC3 have been reported in<br />
approximately 75% <strong>of</strong> all familial and 1% <strong>of</strong> all sporadic heterotaxy cases and have been associated with<br />
VACTERL, a constellation <strong>of</strong> malformations phenotypically overlapping heterotaxy. The presence <strong>of</strong> a<br />
polyalanine (polyA) expansion in one patient with VACTERL is <strong>of</strong> particular interest as pathogenic expansions<br />
have been identified in several other developmentally critical transcription factors, including ZIC2. To further<br />
define the incidence and functional significance <strong>of</strong> ZIC3 mutations in heterotaxy, coding regions and splice<br />
junctions were screened in 200 unrelated heterotaxy patients. Nine mutations (8 novel) were identified,<br />
including a single alanine expansion (c.insGCC159-160). Functional analyses were supplemented by 4<br />
additional recently reported ZIC3 mutations. Aberrant ZIC3 cytoplasmic localization was observed for mutations<br />
spanning multiple nuclear localization domains between amino acids 155 and 318 and correlated with<br />
decreased transactivation <strong>of</strong> a luciferase reporter. A missense mutation within zinc finger 5 (p.A447G)<br />
surprisingly increased luciferase transactivation, despite elevated levels <strong>of</strong> cytoplasmic ZIC3. Neither polyA tract<br />
expansion differed significantly from wildtype with respect to either luciferase transactivation or ZIC3 subcellular<br />
localization. These analyses collectively indicate a higher than expected percentage <strong>of</strong> ZIC3 mutations in<br />
patients with sporadic heterotaxy and suggest alternative pathogenesis <strong>of</strong> some ZIC3 mutations, notably those<br />
within the polyA tract.<br />
23
S1.4 The Genetic Basis <strong>of</strong> Isolated Tetralogy <strong>of</strong> Fallot<br />
Marcel Grunert 1,2,* , Cornelia Dorn 1,2,3,* , Markus Schueler 1,2 , Ilona Dunkel 1 , Jenny Schlesinger 2 , Siegrun Mebus 4 ,<br />
Katherina Bellmann 2,3 , Vladimir Alexi-Meskishvili 5 , Sabine Klaassen 6 , Katharina Wassilew 7 , Bernd<br />
Timmermann 8 , Roland Hetzer 5 , Felix Berger 4 , Silke R. Sperling 1,2,3<br />
1 Group <strong>of</strong> Cardiovascular Genetics, Max Planck Institute for Molecular Genetics, 2 Department <strong>of</strong> Cardiovascular<br />
Genetics, Experimental and Clinical Research Center, Charité – <strong>University</strong> Medicine Berlin and Max Delbrück<br />
Center for Molecular Medicine, 3 Department <strong>of</strong> Biology, Chemistry and Pharmacy at Freie Universität <strong>of</strong> Berlin,<br />
4 Department <strong>of</strong> Pediatric Cardiology, 5 Department <strong>of</strong> Cardiac Surgery and 7 Department <strong>of</strong> Pathology at German<br />
Heart Center Berlin, 6 National Registry <strong>of</strong> Congenital Heart Disease, 8 Next Generation Service Group at Max<br />
Planck Institute for Molecular Genetics, * shared authorship<br />
Background<br />
Tetralogy <strong>of</strong> Fallot (TOF) represents 10% <strong>of</strong> congenital heart disease, which are the most common birth defect<br />
in human. The majority <strong>of</strong> TOF are isolated non-syndromic cases <strong>of</strong> unknown precise cause, which applies for<br />
the majority <strong>of</strong> congenital heart disease.<br />
Methods<br />
We performed targeted resequencing <strong>of</strong> over 1,000 genes and microRNAs as well as whole transcriptome and<br />
miRNome analysis in TOF cases, parents and controls using next-generation sequencing techniques. We<br />
defined a set <strong>of</strong> TOF genes with deleterious variations and which are mutated at a higher rate in TOF subjects<br />
compared to healthy controls.<br />
Results<br />
We identified an oligogenic architecture underlying TOF, which discriminate TOF cases from controls. On<br />
average, TOF subjects show a combination <strong>of</strong> novel and inherited variations in five genes. The majority <strong>of</strong><br />
genes has known association with human cardiac disease and/or shows a cardiac phenotype in mouse mutants.<br />
Seven genes are novel and have not yet been linked to a cardiac phenotype. Functionally interacting yet<br />
individual mutations lead to the same phenotypic outcome during development. The majority <strong>of</strong> TOF genes<br />
shows continuous relevance during adulthood.<br />
Conclusions<br />
Isolated TOF is a genetic disorder involving multiple genes. Although subjects show a range <strong>of</strong> individual<br />
mutations, the phenotypic outcome is similar because TOF genes show common patterns <strong>of</strong> functional<br />
interactions. Sequencing approaches can help to define a genetic risk pr<strong>of</strong>ile for families that can also be used<br />
to define differences in the long-term clinical outcome, which should permit a personalization <strong>of</strong> diagnostic and<br />
therapeutic strategies.<br />
24
S1.5 An Excess <strong>of</strong> Deleterious Variants in VEGF-A Pathway Genes in Down Syndrome-Associated<br />
Atrioventricular Septal Defects<br />
Christine Ackerman 1 , Adam E. Locke 2,8 , Eleanor Feingold 3 , Benjamin Reshey 1 , Karina Espana 1 , Janita<br />
Thusberg 4 , Sean Mooney 4 , Lora J. H. Bean 2 , Kenneth J. Dooley 5 , Clifford Cua 6 , Roger H. Reeves 7 , Stephanie L.<br />
Sherman 2 , Cheryl L. Maslen 1,*<br />
1 Division <strong>of</strong> Cardiovascular Medicine and the Heart Research Center, Oregon Health & Science <strong>University</strong>,<br />
Portland, OR 97239<br />
2 Department <strong>of</strong> Human Genetics, Emory <strong>University</strong>, Atlanta, GA 30033<br />
3 Department <strong>of</strong> Human Genetics, Graduate School <strong>of</strong> Public Health, <strong>University</strong> <strong>of</strong> Pittsburgh, Pittsburgh PA,<br />
15261<br />
4 Buck Institute for Research on Aging, Novato, CA 94945<br />
5 Sibley Heart Center Cardiology, Children’s Hospital <strong>of</strong> Atlanta, Atlanta, GA 30033<br />
6 Heart Center, Nationwide Children’s Hospital, Columbus, OH 43205<br />
7 Department <strong>of</strong> Physiology and the Institute for Genetic Medicine, Johns Hopkins <strong>University</strong> School <strong>of</strong> Medicine,<br />
Baltimore, MD 21205<br />
8 Current address: Center for Statistical Genetics, Department <strong>of</strong> Biostatistics, <strong>University</strong> <strong>of</strong> Michigan School <strong>of</strong><br />
Public Health, Ann Arbor, MI 48109<br />
About half <strong>of</strong> people with trisomy 21 have a congenital heart defect (CHD) while the remainder have a<br />
structurally normal heart, demonstrating that trisomy 21 is a significant risk factor but is not causal for abnormal<br />
heart development. We used a candidate gene approach in a study cohort <strong>of</strong> individuals with Down syndrome<br />
(DS) to determine if rare genetic variants in genes involved in atrioventricular valvuloseptal morphogenesis<br />
contribute to atrioventricular septal defects (AVSD) in this sensitized population. We found a significant excess<br />
(p
S1.6 Identification <strong>of</strong> Novel Mutations in a Large Family with Different Forms <strong>of</strong> Congenital Heart<br />
Disease<br />
AC Fahed,* RC Tanos, † IG El-Rassy, † FF Bitar,† JG Seidman,* CE Seidman* , GM Nemer,†<br />
*Harvard Medical School, Boston, MA<br />
†American <strong>University</strong> <strong>of</strong> Beirut, Beirut, Lebanon<br />
Background: Congenital Heart Disease (CHD) affects more than 1% <strong>of</strong> newborns and is widely believed to be<br />
due to mutations in genes involved in cardiac development. However, to date only about 5% <strong>of</strong> the genetic<br />
causes <strong>of</strong> CHD is known.<br />
Objective: Our objective is to study families with CHD from Lebanon, a highly consanguineous population,<br />
using a combination <strong>of</strong> Sanger and Next-Generation sequencing, as well as SNP genotyping.<br />
Methods: Target-capture sequencing <strong>of</strong> cardiac-enriched genes was performed on more than 150 families with<br />
CHD. The phenotypes include septal defects (atrial and ventricular), valvular disease (pulmonary stenosis,<br />
aortic stenosis, tricuspid atresia, bicuspid aortic valve), coarctation <strong>of</strong> the aorta, tetralogy <strong>of</strong> fallot, transposition<br />
<strong>of</strong> the great arteries, single ventricle, Ebstein anomaly, and atrioventricular canal defects. Mutation-negative<br />
large multiplex families were studied using SNP genotyping and whole-exome sequencing.<br />
Results: Target-capture sequencing analysis <strong>of</strong> 50 families identified several homozygous missense mutations<br />
in cardiac genes (JAG1, NOTCH1, MID1, NF1, NUP188, NFATc4, FLNA). Mutation segregation with the<br />
phenotype was confirmed in most <strong>of</strong> the families. Analysis <strong>of</strong> a particular large family with different forms <strong>of</strong><br />
congenital diseases point out to a multi-factorial mode <strong>of</strong> inheritance involving Hand2, NFATC4, and Nephrin.<br />
Conclusion: Dominant mutations occur in consanguineous populations. The combination <strong>of</strong> SNP genotyping<br />
and next-generation sequencing is an expected method to study gene mutations in families with congenital<br />
heart disease, particularly those with recessive mutations.<br />
26
Platform Session 2: Cardiomyocyte Growth and Regeneration<br />
S2.1 Cardiac hypertrophy in LEOPARD Syndrome is caused by PTPN11 loss-<strong>of</strong>-function activity in the<br />
developing endocardium<br />
Jessica Lauriol 1 , Kimberly Keith 1 , Boding Zhang 3 , Roderick Bronson 2 , Kyu-Ho Lee 3 , and Maria I. Kontaridis 1,4<br />
1 Department <strong>of</strong> Medicine, Division <strong>of</strong> Cardiology, Beth Israel Deaconess Medical Center, Boston, MA.<br />
2 Department <strong>of</strong> Pathology, Harvard Medical School, Boston, MA.<br />
3 Department <strong>of</strong> Cardiovascular Developmental Biology, Medical <strong>University</strong> <strong>of</strong> South Carolina, Charleston, SC.<br />
4 Department <strong>of</strong> Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA.<br />
Mutations in PTPN11, encoding the protein tyrosine phosphatase (PTP) SHP2, cause LEOPARD Syndrome<br />
(LS), an autosomal dominant disorder with multiple cardiac defects, including hypertrophy. Interestingly, LS<br />
mutations are catalytically inactive and behave as loss-<strong>of</strong>-function. However, how LS mutants affect cardiac<br />
development remains unclear.<br />
We generated an inducible “knockin” mouse model expressing the LS-associated Ptpn11 Y279C mutation<br />
(iLS/+). When crossed to EIIA deleter-Cre mice, these (now termed) LS/+ mice recapitulated nearly all aspects<br />
<strong>of</strong> the human LS phenotype, including hypertrophy. To determine whether defects in LS/+ mice could be<br />
attributed directly to aberrant cardiac developmental effects, we examined hearts from WT, LS/+ and LS/LS<br />
embryos at E10.5, E14.5, and E15.5. Both LS/+ and LS/LS developing hearts had significantly diminished<br />
trabeculation at E10.5, as well as valvular hyperplasia and ventricular septal defects (VSD) at E14.5, indicative<br />
<strong>of</strong> defective or delayed cardiac development. In addition, LS/LS embryos had persistent VSD at E15.5 and<br />
cardiac looping defects that resulted in dextraposition <strong>of</strong> the aorta, which led to embryonic lethality between<br />
E14.5 and E15.5.<br />
To determine the lineage-specific effects, we generated neural crest (Wnt1::Cre)-, endothelial (Tie2::Cre)-, and<br />
myocardial (Nkx2.5::Cre)- specific LS/+ expressing mouse lines. Surprisingly, we found that only the<br />
endocardial-specific LS-expressing mice could completely recapitulate the embryonic cardiac developmental<br />
defects observed in the LS/+ phenotype. Moreover, these mice also developed the adult-onset hypertrophy, as<br />
observed in adult LS/+ mice. Taken together, our data indicate that the adult-onset cardiac hypertrophy<br />
associated with LS is caused by PTPN11 loss-<strong>of</strong>-function effects that occur in the developing endocardium.<br />
27
S2.2 Epigenetic regulation <strong>of</strong> cardiac hypertrophy<br />
Qing-Jun Zhang 1 , David Maloney 2 , Anton Simeonov 2 , and Zhi-Ping Liu 1<br />
1 Department <strong>of</strong> Internal medicine, division <strong>of</strong> Cardiology, UT Southwestern<br />
2 National Institutes <strong>of</strong> Health Chemical Genomics Center, National Human Genome Research Institute.<br />
One <strong>of</strong> the major challenges in managing and treating heart failure patients is to develop disease-modifying<br />
drugs that can prevent, reverse, or slow down the disease progression. Upon pathological insults, the heart<br />
undergoes remodeling processes, including left ventricular hypertrophy and reprogramming <strong>of</strong> gene expression.<br />
Understanding the mechanisms involved could provide a key to develop interventional therapeutics. Epigenetic<br />
modification <strong>of</strong> chromatin, including histone methylation, regulates gene transcription in response to<br />
environmental signals. JMJD2A is a trimethyl-lysine specific histone lysine demethylase. To study the role <strong>of</strong><br />
JMJD2A, we generated heart specific JMJD2A overexpression and deletion mouse lines. Our studies with these<br />
genetically modified mice indicated that JMJD2A is required for pathological cardiac hypertrophy. Furthermore,<br />
we show that the demethylase activity <strong>of</strong> JMJD2A is required for its transcriptional activity. To test whether<br />
inhibition <strong>of</strong> JMJD2A enzymatic activity suppresses hypertrophic response, we identified several small molecule<br />
inhibitors <strong>of</strong> JMJD2A. These small molecule inhibitors <strong>of</strong> JMJD2A inhibited the phenylephrine-stimulated<br />
cardiomyocyte hypertrophy in vitro. Our data suggests that JMJD2A enzymatic activity may act as a<br />
hypertrophic determinant and may be an innovative drug target for prevention and treatment <strong>of</strong> pathological<br />
cardiac hypertrophy and heart failure.<br />
28
S2.3 Regulation <strong>of</strong> striated muscle gene expression: A role for Prox1 during cardiac and skeletal<br />
muscle development<br />
Louisa K. Petchey 1 , Paul R. Riley 1<br />
1 Molecular Medicine Unit, UCL Institute <strong>of</strong> Child Health, London WC1N 1EH, UK<br />
Is<strong>of</strong>orm-specific expression <strong>of</strong> muscle structural genes is a crucial component <strong>of</strong> the structural, functional and<br />
metabolic distinction that exists between the fibre types comprising striated cardiac and skeletal muscle. The<br />
homeobox transcription factor Prox1 has previously been shown to be expressed in slow-twitch skeletal muscle<br />
in zebrafish and to be required for normal cardiac muscle development in mice. Using Nkx2.5-Cre, Myf5-Cre<br />
and Prox1 flox mouse lines to knockout Prox1 in both striated muscle types, we can identify for the first time a role<br />
for Prox1 in the repression <strong>of</strong> three key fast-twitch skeletal muscle genes, Tnnt3, Tnni2 and Myl1, in both<br />
cardiac and slow-twitch skeletal muscle. Prox1 has an additional role in skeletal muscle in the regulation <strong>of</strong><br />
myosin heavy chain is<strong>of</strong>orm expression. This implicates Prox1 in a complex regulatory network that determines<br />
fibre type in skeletal muscle, where it functions downstream <strong>of</strong> Sox6 and upstream <strong>of</strong> Six1; and characterises a<br />
novel role for Prox1 during heart development. The inappropriate expression <strong>of</strong> fast-twitch skeletal muscle<br />
genes is known to negatively impact cardiac function while some human cardiac and skeletal myopathies have<br />
been associated with incorrect is<strong>of</strong>orm expression <strong>of</strong> structural genes. New opportunities for intervening in the<br />
molecular mechanisms that underlie these pathologies will be gained from a better understanding <strong>of</strong> their<br />
developmental regulation.<br />
29
S2.4 CIP, a novel cardiac nuclear protein regulates cardiac function and remodeling<br />
Zhan-Peng Huang, Hee Young Seok, Jinghai Chen, and Da-Zhi Wang<br />
Department <strong>of</strong> Cardiology Children's Hospital Boston Harvard Medical School 320 Longwood Avenue Boston,<br />
MA 02115<br />
Mammalian heart has minimal regenerative capacity. In response to mechanical or pathological stress, the heart<br />
undergoes cardiac remodeling. Pressure and volume overload in the heart cause increased size (hypertrophic<br />
growth) <strong>of</strong> cardiomyocytes. Whereas the regulatory pathways that activate cardiac hypertrophy have been well<br />
established, the molecular events that inhibit or repress cardiac hypertrophy are less known. Here, we report the<br />
identification, characterization and functional examination <strong>of</strong> CIP, a novel cardiac Isl1-interacting protein. CIP<br />
was identified from a bioinformatic search for novel cardiac-expressed genes in mouse embryonic hearts. CIP<br />
encodes a nuclear protein without recognizable motifs. Northern blotting, in situ hybridization and reporter gene<br />
tracing demonstrated that CIP is highly expressed in cardiomyocytes <strong>of</strong> developing and adult hearts. Yeast-twohybrid<br />
screening identified Isl1, a LIM/homeodomain transcription factor essential for the specification <strong>of</strong> cardiac<br />
progenitor cells in the second heart field, as a co-factor <strong>of</strong> CIP. CIP directly interacted with Isl1 and we mapped<br />
the domains <strong>of</strong> these two proteins which mediate their interaction. We show that CIP represses the<br />
transcriptional activity <strong>of</strong> Isl1 in the activation <strong>of</strong> the MEF2C enhancer. The expression <strong>of</strong> CIP was dramatically<br />
reduced in hypertrophic cardiomyocytes. Most importantly, overexpression <strong>of</strong> CIP repressed agonist-induced<br />
cardiomyocyte hypertrophy. Interestingly, recent work showed that CIP also interacts with Lamin A/C (LMNA), a<br />
causative gene mutated in patients with DCM and other disorders. We found that the expression <strong>of</strong> CIP was<br />
markedly regulated in animal models <strong>of</strong> cardiac diseases, including HCM and DCM. Furthermore, our<br />
preliminary data showed that CIP knockout mice display impaired cardiac function. Together, our studies identify<br />
CIP a novel regulator <strong>of</strong> cardiac remodeling and disease.<br />
30
S2.5 Yap1 regulates cardiomyocyte proliferation through PIK3CB<br />
Zhiqiang Lin(a), Alexander von Gisea(a), Pingzhu Zhou(a) Jessie Buck(a) William Pu(a,b)<br />
(a)Department <strong>of</strong> Cardiology and (b) Stem Cell Program, Children’s Hospital Boston, Boston, MA 02115<br />
Cardiomyocyte loss is a major underlying cause <strong>of</strong> heart failure. Works focusing on cardiomyocyte proliferation<br />
regulation have shown that adult cardiomyocytes do proliferate to a limited extent, but not enough to show any<br />
observable clinical benefits. Efforts are needed to deciphering genes capable <strong>of</strong> driving cardiomyocytes to reenter<br />
cell cycle. The Hippo kinase cascade is an important regulator <strong>of</strong> organ growth, including heart. A major<br />
target <strong>of</strong> this kinase cascade is YAP1. Our data showed that fetal Yap1 inactivation caused marked, lethal<br />
myocardial hypoplasia and decreased cardiomyocyte proliferation, whereas fetal activation <strong>of</strong> YAP1 stimulated<br />
cardiomyocyte proliferation. Remarkably, YAP1 activation was sufficient to stimulate proliferation <strong>of</strong> postnatal<br />
cardiomyocytes, both in culture and in the intact heart. To characterize the mechanism <strong>of</strong> YAP1 induced<br />
cardiomyocyte proliferation, we carried out microarray and Chip sequencing assays. By combining the<br />
microarray da ta and the Chip sequencing results, we found several genes that possibly regulated by YAP1.<br />
PIK3CB was validated to be a direct target <strong>of</strong> Yap1, and a regulatory element residing in the first intron <strong>of</strong><br />
PIK3CB was characterized. Knocking down <strong>of</strong> PIK3CB significantly reduced the YAP1 induced cardiomyocyte<br />
proliferation, however, TGX221, a specific PIK3CB kinase inhibitor failed to weaken the effects <strong>of</strong> YAP1 on<br />
cardiomyocyte proliferation. Overexpression <strong>of</strong> either PIK3CB, or PIK3CBK805R, a kinase dead mutation<br />
version, was sufficient to drive neonatal cardiomyocytes to re-enter cell cycle in vitro. These studies<br />
demonstrate that YAP1 is a crucial regulator <strong>of</strong> cardiomyocyte proliferation, and YAP1 regulates cardiomyocyte<br />
proliferation partially through PIK3CB.<br />
31
S2.6 A Transitional Extracellular Matrix Instructs Epicardial-mediated Vertebrate Heart Regeneration<br />
Sarah Mercer 1 , Claudia Guzman 1 , Chia-ho Cheng 2 , Shannon Odelberg 3 , Ken Marx 2 and Hans-Georg Simon 1<br />
1<br />
Department <strong>of</strong> Pediatrics and Children’s Memorial Research Center, Northwestern <strong>University</strong> Feinberg School<br />
<strong>of</strong> Medicine, <strong>Chicago</strong> IL, USA<br />
2<br />
Department <strong>of</strong> Chemistry, <strong>University</strong> <strong>of</strong> Massachusetts-Lowell, USA<br />
3<br />
Department <strong>of</strong> Internal Medicine, <strong>University</strong> <strong>of</strong> Utah School <strong>of</strong> Medicine, Salt Lake City UT, USA<br />
Unlike humans, certain vertebrates including newts and zebrafish possess extraordinary abilities to functionally<br />
regenerate lost appendages and injured organs, including cardiac muscle. We aim to discover the underpinning<br />
mechanisms that regulate complex tissue rebuilding in these regeneration-competent species, ultimately<br />
developing strategies for regenerative wound healing in mammals. Here, we present new evidence that the<br />
extracellular matrix (ECM) provides signals to tissues and cells essential for the induction and maintenance <strong>of</strong><br />
regenerative processes in cardiac muscle. Comprehensive mining <strong>of</strong> DNA microarrays <strong>of</strong> regenerating newt and<br />
zebrafish hearts with data comparison to the scar-forming response following myocardial infarction in mice and<br />
humans provided a signature <strong>of</strong> conserved regenerative gene activities. Differential expression and Gene<br />
Ontology analyses revealed that distinct ECM components and ECM-modifying proteases are among the<br />
earliest upregulated and most significantly enriched genes in response to injury in both the newt and zebrafish.<br />
In contrast, following mammalian cardiac injury, immune and inflammatory responses are significantly activated<br />
instead <strong>of</strong> ECM genes. Complementary immunohistochemistry studies in the newt demonstrated dynamic<br />
spatial and temporal changes in ECM composition between normal and regenerating heart tissues, especially in<br />
the epicardium early in the regenerative response. We show in vivo and under defined culture conditions that<br />
the proliferative and migratory response <strong>of</strong> cardiomyocytes is directly linked to distinct matrix remodeling in both<br />
the myocardium and epicardium <strong>of</strong> the regenerating newt heart. Collectively, these results provide a novel<br />
understanding <strong>of</strong> the regenerative process, suggesting that an evolutionarily conserved, regeneration-specific<br />
matrix instructs cell activities essential for cardiac muscle regeneration.<br />
32
Platform Session 3: Genomics and Gene Regulation<br />
S3.1 Isolation <strong>of</strong> nuclei from specific cardiac lineages in zebrafish for genome-wide and epigenomic<br />
analyses<br />
Bushra Gorsi and H. Joseph Yost<br />
Department <strong>of</strong> Neurobiology and Anatomy, <strong>University</strong> <strong>of</strong> Utah Molecular Medicine Program, Eccles Institute <strong>of</strong><br />
Human Genetics, Building 533, Room 3160, 15 North 2030 East, Salt Lake City, Utah 84112-5330, USA.<br />
To understand the critical gene regulatory interactions that drive atrial versus ventricle identity, we are using<br />
genome-wide analysis <strong>of</strong> differentiating cardiomyocytes to uncover changes in epigenetic and transcriptome<br />
pr<strong>of</strong>iles. Although advances in sequencing technology have greatly enhanced our ability to characterize<br />
genome-wide changes in epigenetic modifications and gene expression, this has been constrained by the<br />
technical challenges faced in purifying specific cell lineages in adequate amounts from developing systems.<br />
Therefore we are developing a novel approach that will allow us to isolate nuclei <strong>of</strong> differentiating<br />
cardiomyocytes in-vivo.<br />
We have created transgenic lines in which a biotin ligase recognition peptide (BLRP) is fused to an outer<br />
membrane nuclear envelope protein (NEP). Regulated expression <strong>of</strong> BirA enzyme drives the in vivo biotinylation<br />
<strong>of</strong> BLRP-NEP, allowing purification <strong>of</strong> nuclei with streptavidin-conjugated beads. When BirA is ubiquitously<br />
expressed, we can successfully elute chromatin threefold higher from transgenic embryos with biotinylated<br />
nuclei than transgenic embryos devoid <strong>of</strong> BirA enzyme. We successfully performed ChIP with multiple histone<br />
marks, indicating that the chromatin within these purified nuclei is useful for epigenomic analyses. We are<br />
creating double transgenics in which BLRP-NEP is biotinylated in specific heart lineages in order to capture<br />
chromatin and nuclear RNA (mRNA and miRNA) from cardiac progenitors, ventricle precursors and atrial<br />
precursors. This will allow us to perform genome-wide analysis <strong>of</strong> epigenetic and transciptome pr<strong>of</strong>iles in<br />
specific subsets <strong>of</strong> lineages and tissues during any stage <strong>of</strong> zebrafish cardiac development.<br />
Supported in part by U01HL098179 (NHLBI Bench-to-Bassinet program)<br />
33
S3.2 TU-tagging in mice defines dynamic gene expression programs in specific cardiovascular or other<br />
cell types within intact tissues<br />
Leslie Gay 1-3 , Michael R. Miller 1-3 , Vidusha Devasthali 2 , P. Brit Ventura 2 , Zer Vue 4 , Heather L. Thompson 4 , Sally<br />
Temple 5 , Hui Zong 2 , Michael D. Cleary 4 , Kryn Stankunas 2 , Chris Q. Doe 1-3<br />
Institute <strong>of</strong> Neuroscience 1 , Institute <strong>of</strong> Molecular Biology 2 , Howard Hughes Medical Institute 3 , <strong>University</strong> <strong>of</strong><br />
Oregon, Eugene, OR 97403<br />
4<br />
Division <strong>of</strong> Natural Sciences, <strong>University</strong> <strong>of</strong> California, Merced, CA 95340<br />
5<br />
Neural Stem Cell Institute, Rensselaer, NY 12144<br />
Changes in gene expression programs underlie all aspects <strong>of</strong> cardiovascular development and disease.<br />
Therefore, transcriptional pr<strong>of</strong>iling is a powerful and direct approach to study these and other biological<br />
processes. Current methods are limited by difficulties isolating specific cell populations from complex tissues,<br />
changes in gene expression that occur during sample preparation, and the inability to distinguish dynamic<br />
transcriptional changes within a cellular pool <strong>of</strong> pre-existing RNA. To overcome these limitations in mouse<br />
research, we have developed a method called “TU-tagging” that allows for purification <strong>of</strong> newly transcribed RNA<br />
from defined cell types. With TU-tagging, genetic and chemical methods intersect to provide spatiotemporal<br />
controlled RNA labeling in vivo. Cre-induced expression <strong>of</strong> UPRT using a newly-developed transgenic mouse<br />
(CA:loxStoploxUPRT) directs the incorporation <strong>of</strong> injected 4-thiouracil (4TU) into nascent RNA, generating a<br />
pulse <strong>of</strong> cell type-specific thiolated RNA. The thio-RNA is readily purified from whole tissue RNA preparations<br />
and RNA-seq is used to define “acute” transcriptomes. We validate mouse TU-tagging by using<br />
Tie2:Cre;CA:loxStoploxUPRT double transgenic mice to purify endothelial RNA from brain or<br />
endothelial/endocardial RNA from heart at both embryonic and postnatal stages. In each case, the vast majority<br />
<strong>of</strong> the most enriched transcripts are verified as being expressed in Tie2:Cre marked cell lineages. We define a<br />
core set <strong>of</strong> pan-endothelial transcripts and identify groups <strong>of</strong> endothelial/endocardial lineage transcripts with<br />
shared specific expression patterns. TU-tagging provides a novel, sensitive method for characterizing<br />
transcriptome dynamics in rare cell types within the intact mouse.<br />
34
S3.3 Escape <strong>of</strong> Gene Body DNA Methylation in Cardiac Genes is Cooperative with the Cell Type-specific<br />
Expression Patterns<br />
Mayumi Oda, Shinji Makino, Hirokazu Enomoto, Ruri Kaneda, Shinsuke Yuasa and Keiichi Fukuda<br />
Center for Integrated Medical Research, School <strong>of</strong> Medicine, Keio <strong>University</strong>, Tokyo 160-8582 JAPAN.<br />
Mammalian genome is highly methylated in C-G dinucleotides (CpGs), over 60% <strong>of</strong> which are believed to be<br />
methylated. The effect <strong>of</strong> DNA methylation has been investigated intensively in the promoter regions, but it has<br />
still been unknown in non-promoter regions, especially in the cell type-specific effect. Although we previously<br />
reported that gene body DNA methylation levels are correlated with the transcription levels and the replication<br />
timing in human cell lines (Suzuki et al. Genome Res 2011), function <strong>of</strong> gene body DNA methylation levels in<br />
developing cells is unknown. In this study, we focused on the DNA methylation pattern in cardiomyocytes as the<br />
established differentiated cells. We examined genome-wide DNA methylation pr<strong>of</strong>iles <strong>of</strong> cardiomyocytes and<br />
non-cardiomyocyte cells from different developmental stages by the deep sequencing technique, and found that<br />
the gene body regions <strong>of</strong> some cardiac genes, including myosin heavy chain (MHC) genes, are less methylated<br />
than other genes, where the DNA methylation levels were dramatically decreased in the latter half <strong>of</strong> pregnancy.<br />
To evaluate the effect <strong>of</strong> gene body DNA methylation, we performed luciferase reporter assay by the<br />
reconstruction using the SssI-methylated and unmethylated gene body fragments, and confirmed that the gene<br />
body DNA methylation is repressive in the gene expression, mostly at the transcriptional level. Collectively, the<br />
gene body DNA methylation levels in some cardiac genes were developmentally regulated and possibly<br />
enhance the transcription <strong>of</strong> cardiac genes.<br />
35
S3.4 The novel mechanism <strong>of</strong> histone-chromatin regulation for cardiomyocytes regeneration<br />
Ryo Nakamaura, 1 Kazuko Koshiba-Takeuchi, 1 Yuko Tsukahara, 1 Tetsuo Sasano, 3 Mizuyo Kojima, 1 Tetsushi<br />
Furukawa, 3 Hesham A Sadek, 4 Jun K Takeuchi, 1,2<br />
1 Univ <strong>of</strong> Tokyo, 2 JST PRESTO, 3 Tokyo Medical and Dental Univ, 4 Univ <strong>of</strong> Texas Southwestern Medical Center<br />
Fishes and amphibians have high capacities for heart regeneration even in the adult, whereas we mammalians<br />
lose such regeneration capacities. Understanding the mechanism <strong>of</strong> heart regeneration should provide the most<br />
important cues on the therapies for human heart failure. We found that SWI/SNF-BAF type cardiac chromatin<br />
remodeling factors and histone regulators were strongly up-regulated within 12 hours after resection <strong>of</strong> ventricle<br />
both in neonatal mice and axolotl, and these expression was maintained for one week during regeneration. To<br />
address whether these factors function as the early response factors in mammalian heart regeneration, we<br />
constructed BAF-overexpression (BAF-TG) mice, which showed stable expression <strong>of</strong> BAFs even in the adult<br />
heart. Interestingly, BAF-TG prevented fibrosis post myocardial infarction and regenerated their injured parts<br />
with the proliferation <strong>of</strong> cardiomyocytes. In vivo ChIP analyses revealed that the SWI/SNF core factor, Brg1,<br />
directly regulated several cardiac fetal genes’ promoters such as Nppa, Tnnt2, Myl7 only at the embryonic<br />
stages. Surprisingly, in the BAF-TG, major cardiac fetal genes’ promoters were still opened in the adult state.<br />
And si-treatment <strong>of</strong> polycomb or repress-type chromatin factor s increased cell proliferation in vitro. These data<br />
demonstrate that histone-chromatin conformation is changed depending on the developmental stages on<br />
several cardiac genes relating with the regeneration capacity.<br />
36
S3.5 Epigenetic regionalization <strong>of</strong> the developing mouse heart<br />
Scott Smemo*, Noboru J. Sakabe*, Ivy Aneas, Marcelo A. Nobrega<br />
Department <strong>of</strong> Human Genetics, <strong>University</strong> <strong>of</strong> <strong>Chicago</strong>, <strong>Chicago</strong>, IL.<br />
A multitude <strong>of</strong> differences in gene expression have been described and correlated with cardiac chamber identity,<br />
structure, function and disease. Nonetheless, it is not fully resolved how these transcriptional spatial<br />
asymmetries, evident at even the very early stages <strong>of</strong> morphogenesis, are established and maintained. An<br />
intriguing hypothesis is that these asymmetries are contributed to by differences in chromatin accessibility,<br />
which potentiate or exclude expression <strong>of</strong> specific genes. By performing transcriptional pr<strong>of</strong>iling by RNA-seq on<br />
isolated left and right ventricles <strong>of</strong> embryonic mice and correlating the findings with ventricular ChIP-seq data for<br />
H3K27me3, a mark <strong>of</strong> closed chromatin associated with gene repression, and H3K4me1, a histone modification<br />
associated with open chromatin and active genes, we observe a significant subset <strong>of</strong> chamber-specific cardiac<br />
genes, including Tbx5, that are selectively marked by both H3K4me1 and H3K27me3 in opposite chambers. In<br />
the left ventricle, these genes are functionally linked to roles in energy metabolism, whereas in the right<br />
ventricle, coordinately marked genes are associated with developmental roles. We demonstrate that Tbx5<br />
enhancers have the potential <strong>of</strong> driving expression in broad cardiac domains, and that the restricted Tbx5<br />
expression is mainly due to regionalization <strong>of</strong> epigenetic marks. Furthermore, this spatially regionalized pattern<br />
<strong>of</strong> gene repression is redeployed to limit Tbx5 expression to forelimb and not hindlimb, highlighting a general<br />
chromatin-based mechanism to affect spatially-restricted expression. Beyond our general understanding <strong>of</strong> how<br />
the developing heart is patterned, these findings have implications for efforts at directed differentiation <strong>of</strong> stem<br />
cells into cardiomyocytes for therapeutic uses.<br />
37
S3.6 Drosophila microRNA-1 Genetically Interacts with little imaginal discs, a histone demethylase.<br />
Isabelle N. King 1,2<br />
1 Gladstone Institute <strong>of</strong> Cardiovascular Disease, 1650 Owens Street, San Francisco, CA 94158, 2 Department <strong>of</strong><br />
Pediatrics, <strong>University</strong> <strong>of</strong> California, San Francisco, San Francisco, CA, 94143.<br />
The expression pattern and function for the microRNA miR-1 is conserved in both invertebrates and vertebrates,<br />
suggesting that it modulates some <strong>of</strong> the most fundamental processes within cardiac and skeletal muscle,<br />
including Notch signaling. We performed a forward genetic screen in Drosophila that identified little imaginal<br />
discs (lid), a chromatin-remodeling enzyme, as a genetic partner <strong>of</strong> dmiR-1. Lid demethylates histone H3 lysine<br />
4 (H3K4), thereby epigenetically decreasing the activity <strong>of</strong> selected promotors. Lid forms a complex with Notch<br />
pathway regulatory proteins, and loss <strong>of</strong> lid activity results in de-repression <strong>of</strong> Notch downstream targets. We<br />
have found that in flies, heterozygosity <strong>of</strong> lid in the context <strong>of</strong> dmiR-1 overexpression resulted in exacerbation <strong>of</strong><br />
the dmiR-1 overexpression phenotype, consistent with de-repression <strong>of</strong> Notch signaling. Furthermore, we<br />
discovered that use <strong>of</strong> a balancer chromosome containing a mutation in the Notch ligand Serrate, unmasked an<br />
indirect flight muscle phenotype in lid (-/-) flies that was modified upon manipulation <strong>of</strong> dmiR-1 levels. These<br />
results imply that dmiR-1 may selectively modulate the epigenetic modification <strong>of</strong> histones at Notch-responsive<br />
loci during fly heart and muscle development.<br />
38
Platform Session 4: Neural Crest and Vascular Development<br />
S4.1 Early neural crest-restricted Noggin over-expression results in congenital crani<strong>of</strong>acial and<br />
cardiovascular defects<br />
Zachary Neeb, Paige Snider and Simon J. Conway<br />
Program in Developmental Biology and Neonatal Medicine, Herman B Wells Center for Pediatric Research,<br />
Indiana <strong>University</strong> School <strong>of</strong> Medicine, Indianapolis, IN 46202.<br />
Noggin is a secreted BMP antagonist that binds BMP ligands and prevents their activation <strong>of</strong> receptors. Noggin<br />
(in concert with Wnts, FGFs, Delta/Notch) plays a key upstream role in neural crest cell (NCC) induction by<br />
generating a BMP morphogenic gradient that activates a cascade <strong>of</strong> transcription factors culminating in the<br />
ultimate steps <strong>of</strong> NCC differentiation. NCC derivatives are absolutely essential for cardiac outflow tract (OFT)<br />
remodeling, specifically during OFT septation and patterning <strong>of</strong> the great vessels that exit the heart.<br />
Using Cre/loxP, constitutive Noggin expression was induced within NCC via Pax3 Cre (very early within neural<br />
tube), Wnt1-Cre (early within neural tube) or Peri-Cre (post-migratory) drivers. Consistent with its in vitro role in<br />
negatively regulating BMP signaling, Westerns confirmed Noggin overexpression significantly suppressed<br />
phosphorylation levels <strong>of</strong> Smad1/5/8 but left Smad2/3 unaffected in vivo. Moreover, Wnt1-Cre and Pax3 Cre -<br />
mediated suppression <strong>of</strong> BMP signaling resulted in subsequent loss <strong>of</strong> NCC-derived crani<strong>of</strong>acial, pharyngeal<br />
and OFT cushion tissues. Increased cell death was observed in pharyngeal arch NCC and DRG (although cell<br />
proliferation was unaltered), but not within the OFT itself. Lineage mapping demonstrated that NCC emigration<br />
was not affected in either Pax3 Cre ;Noggin or Wnt1-Cre;Noggin mutants, but that subsequent colonization <strong>of</strong> the<br />
OFT was significantly reduced in Wnt1-Cre;Noggin and completely absent in Pax3 Cre ;Noggin mutants. Further,<br />
although Wnt1-Cre;Noggin mutants are viable until birth, Pax3 Cre ;Noggin mutants all die by E14. Conversely,<br />
Peri-Cre mediated suppression <strong>of</strong> BMP signaling in post-migratory NCC had no effect and mutants are viable at<br />
birth. Taken together, these data show that conditional BMP inhibition within the NCC may exhibit different<br />
effects dependent upon timing and that tightly regulated TGFβ superfamily signaling plays an essential role<br />
during crani<strong>of</strong>acial and cardiac NCC survival in vivo.<br />
39
S4.2 The Role <strong>of</strong> Fibronectin - Integrin Signaling in Pharyngeal Microenvironment in Modulation <strong>of</strong><br />
Cardiac Neural Crest Cell Development<br />
Dongying Chen and Sophie Astr<strong>of</strong><br />
Thomas Jefferson <strong>University</strong>, Philadelphia, USA<br />
Patterning <strong>of</strong> Pharyngeal Arch Arteries (PAAs) is a critical process in embryonic cardiovascular development.<br />
Alterations that perturb this process cause cardiovascular abnormalities such as human DiGeorge Syndrome.<br />
Our lab previously discovered defective development <strong>of</strong> CNCCs in global fibronectin (FN)-null or integrin a5-null<br />
mutants. In order to determine the roles <strong>of</strong> FN synthesized by the pharyngeal microenvironment during CNCC<br />
development, we conditionally ablated FN or the α5 subunit <strong>of</strong> its integrin receptor using Isl1 Cre strain <strong>of</strong> mice.<br />
We found that conditional inactivation <strong>of</strong> FN or integrinα5 using Isl1 Cre cause embryonic/neonatal lethality. Both<br />
<strong>of</strong> these conditional mutants display various cardiovascular and glandular anomalies associated with defective<br />
PAA and CNCC development. Moreover, histological analyses reveal defective development <strong>of</strong> ventricular<br />
myocardium and muscular ventricular septum, phenotypes dependent on the proper development <strong>of</strong> the<br />
mesodermal cardiac progenitors. Taken together, these phenotypes suggest that FN-integrin α5 signaling to<br />
distinct tissues comprising pharyngeal microenvironment play critical roles in PAA development by modulating<br />
interactions between CNCCs and non-CNCCs. To understand the basis <strong>of</strong> these phenotypes, we have<br />
investigated the contribution <strong>of</strong> CNCC to PAA formation/remodeling and found that FN promotes survival <strong>of</strong><br />
CNCCs and cardiac progenitors. In addition, FN synthesized by pharyngeal tissues regulates CNCC navigation.<br />
We are investigating possible roles <strong>of</strong> FN and integrin α5-regulated signaling in regulation <strong>of</strong> morphogens,<br />
guidance molecules and growth factor signaling to tissues comprising the pharyngeal microenvironment. Taken<br />
together, our studies provide important insights into how tissue microenvironment regulates morphogenesis <strong>of</strong><br />
complex organs from diverse populations <strong>of</strong> progenitors.<br />
40
S4.3 Slit3-Robo1/2 signalling controls cardiac innervation and ventricular septum development by<br />
regulating neural crest cell survival<br />
Mathilda T.M. Mommersteeg 1 , William D. Andrews 1 , Alain Chédotal 2 , Vincent M. Christ<strong>of</strong>fels 3 , John G.<br />
Parnavelas 1<br />
1 Department <strong>of</strong> Cell and Developmental Biology, <strong>University</strong> College London, London, UK<br />
2 Institut National de la Santé et de la Recherche Médicale, UMR S968, Institut de la Vision, Paris, France<br />
3 Heart Failure Research Center, Department <strong>of</strong> Anatomy, Embryology and Physiology, Amsterdam, The<br />
Netherlands<br />
The Slit-Robo signalling pathway has been shown to have pleiotropic effects in Drosophila heart development,<br />
however, its involvement in mammalian heart formation is yet largely unknown. Here, we analysed the role <strong>of</strong><br />
this signalling pathway during murine heart development. We observed extensive expression <strong>of</strong> both Robo1 and<br />
Robo2 receptors and their ligands, Slit2 and Slit3, in and around the developing heart. Analysis <strong>of</strong> mice lacking<br />
Robo1 revealed membranous ventricular septum defects and decreased cardiac innervation, while animals<br />
lacking Robo2 did not display such defects. However, the combined absence <strong>of</strong> both Robo1 and Robo2 caused<br />
increased incidence and severity <strong>of</strong> membranous ventricular septum defects. Mice lacking the Slit1 and Slit2<br />
ligands did not reveal any abnormalities, but Slit3 mutants recapitulated the membranous ventricular septum<br />
defect. Interestingly, the density <strong>of</strong> cardiac innervation was increased in Slit3 mutants. The combined absence<br />
<strong>of</strong> the membranous ventricular septum and innervation defects suggested a defect in cardiac neural crest<br />
contribution. Detailed cell counts showed a reduction in neural crest cell contribution to the outflow tract. Further<br />
analyses indicated increased apoptosis in the neural crest, just before entering the heart, suggesting that cell<br />
death underlies its reduced contribution to the heart. Our data indicate a novel role for Slit3-Robo1/2 interaction<br />
in cardiac neural crest survival and, thereby, in the formation <strong>of</strong> the membranous ventricular septum and cardiac<br />
innervation.<br />
41
S4.4 The neural crest contributes to coronary artery smooth muscle formation through endothelin<br />
signaling<br />
Yuichiro Arima*, Sachiko Miyagawa-Tomita**, Kazuhiro Maeda**, Koichi Nishiyama*, Rieko Asai*, Ki-Sung<br />
Kim*, Yasunobu Uchijima*, Hisao Ogawa***, Yukiko Kurihara* and Hiroki Kurihara*<br />
*Department <strong>of</strong> Physiological Chemistry and Metabolism, Graduate School <strong>of</strong> Medicine, <strong>University</strong> <strong>of</strong> Tokyo,<br />
Tokyo, Japan.<br />
**Department <strong>of</strong> Pediatric Cardiology, Tokyo Women’s Medical <strong>University</strong>, Tokyo, Japan.<br />
*** Department <strong>of</strong> Cardiovascular Medicine, Faculty <strong>of</strong> Life Sciences, Kumamoto <strong>University</strong>, Kumamoto, Japan.<br />
Endothelin-1 (ET1) /Endothelin A receptor (ETAR) axis has important roles in regulating cardiovascular<br />
homeostasis and organ development. We have previously reported that ET1 and ETAR knockout mice display<br />
crani<strong>of</strong>acial defects and aortic arch malformations. We also found their expressions in the developing coronary<br />
artery smooth muscle cells (CASMCs), however their roles in coronary artery development had been unknown.<br />
Here we show that ET1/ETAR axis insufficiency disturbs coronary artery development. Fetal coronary<br />
angiography revealed some septal branches were abnormally enlarged in both ET-1 and ETAR KO mice at<br />
embryonic day 17.5 (E17.5) and the branching pattern <strong>of</strong> the septal branch was also altered in both KO mice.<br />
Immunohistochemical analysis revealed partial lack <strong>of</strong> CASMCs in KO mice and serial analysis showed these<br />
malformations were occurred at the remodeling stage <strong>of</strong> coronary arteries. Each phenotype appeared in the<br />
septal branch-specific manner, then we hypothesized these segment specificities stood on the variety <strong>of</strong> cell<br />
sources <strong>of</strong> CASMCs. Fate mapping using Wnt1-Cre mice suggested that lacked CASMCs were specifically<br />
derived from the neural crest cells.<br />
To analyze the contribution <strong>of</strong> neural crest cells to CASMCs in detail, we used quail-chick chimera technique<br />
and neural crest ablation models. Quail-chick chimera with a particular region <strong>of</strong> the neural crest recaptured the<br />
preferential distribution pattern to the interventricular septum region. Chick ablation models also showed<br />
coronary artery malformations similar to those in ET-1 and ETAR KO mice. These results indicate novel<br />
contribution <strong>of</strong> the neural crest to coronary artery formation partly through endothelin signaling.<br />
42
S4.5 CASTOR directly regulates a novel Egfl7/RhoA pathway to promote blood vessel development and<br />
morphogenesis<br />
Marta Szmacinski, Kathleen Christine, Nirav Amin, Joan Taylor, Frank L. Conlon<br />
The <strong>University</strong> <strong>of</strong> North Carolina, Chapel Hill<br />
The formation <strong>of</strong> the vascular system is essential for embryonic development and homeostasis. Consistent with<br />
recent genome wide association studies implicating a genetic link between the transcription factor CASTOR<br />
(CST) and high blood pressure and hypertension, we demonstrate here that CST has an evolutionarily<br />
conserved function in blood vessel formation; in the absence <strong>of</strong> CST, Xenopus embryos fail to develop a fully<br />
branched and lumenized vascular system and human endothelial cells (HUVECs) lacking CST display dramatic<br />
defects in adhesion/contractility and cell division. Using chromatin immunoprecipitation (ChIP), we go on to<br />
identify Epidermal growth factor-like domain 7 (Egfl7) as a direct transcriptional target <strong>of</strong> CST. We demonstrate<br />
that CST is required for expression <strong>of</strong> Egfl7, that CST is endogenously bound to the Egfl7 locus in the<br />
developing embryo, and that depletion <strong>of</strong> EGFL7 phenocopies CST depletion in whole embryos and in<br />
HUVECs. We further show that the adhesio n/contractility abnormalities due to depletion <strong>of</strong> CST in HUVECs can<br />
be rescued by the reintroduction <strong>of</strong> EGFL7. Critically, we have demonstrated that CST and EGFL7 modulate<br />
endothelial cell shape and contractility through the small GTPase RhoA. Collectively, these data support a<br />
mechanism whereby CST directly regulates its transcriptional target Egfl7 to promote RhoA-mediated<br />
endothelial cell shape and adhesion changes to establish the vasculature and hence, these studies provide<br />
insight into the cellular and molecular basis for vascular disease associated with the loss <strong>of</strong> CST.<br />
43
S4.6 Nephronectin regulates axial vein morphogenesis in zebrafish<br />
Chinmoy Patra* 1 , Filomena Ricciardi 1 , Iva Nikolic 2 , Mirko HH Schmidt 2 , Benno Jungblut 1 , Felix B. Engel 1<br />
Max-Planck-Institute for Heart and Lung Research<br />
Angiogenesis is the development <strong>of</strong> new vessels from pre-existing vessels. This is a critical morphological event<br />
both in organ development as well as in diseases. Like in other vertebrates, in zebrafish vessels form a complex<br />
network in order to fulfill tissue oxygen demands. Development <strong>of</strong> complex vascular networks is dependent on<br />
the directional migration <strong>of</strong> groups <strong>of</strong> endothelial cells, which is called angiogenic sprouting. Here we have<br />
demonstrated that in zebrafish the extracellular matrix protein, nephronectin, is transiently expressed in the<br />
caudal vein plexus forming region at the time <strong>of</strong> caudal vein sprouting at around 30 hours post fertilization (hpf).<br />
Morpholino-mediated nephronectin depletion resulted in the malformation <strong>of</strong> the caudal vein plexus and the<br />
ventral vein and in the frequent loss <strong>of</strong> inter-segmental veins. Time-lapse analysis from 28 hpf to 40 hpf<br />
indicated a decreased in the frequency <strong>of</strong> caudal vein sprout formation in nephronectin morphants. In addition,<br />
existing sprouting appeared multi-directional indicating a navigation problem. Biochemical analysis<br />
demonstrated that nephronectin is able to bind to the integrin αV/β3 heterodimer. Importantly, integrin αV and<br />
nephronectin expression overlapped in the region <strong>of</strong> the caudal vein plexus. Moreover, morpholino-mediated<br />
integrin αV knockdown in zebrafish phenocopied nephronectin depletion. Taken together, our data indicate that<br />
nephronectin regulates directional sprouting <strong>of</strong> the axial vein in zebrafish, which might be via integrin αV.<br />
44
Platform Session 5: Coronaries, Epicardium, and Conduction System<br />
S5.1 Ets-1 Regulates the Migration <strong>of</strong> Coronary Vascular Precurors and Coronary Endothelial Cell<br />
Proliferation<br />
Gene H. Kim, Saoirse S. McSharry, Zhiguang Gao, Ankit Bhatia, Judy Earley, and Eric C. Svensson<br />
Department <strong>of</strong> Medicine, The Universtiy <strong>of</strong> <strong>Chicago</strong>, <strong>Chicago</strong>, IL 60637<br />
We have recently described a cardiac developmental defect in mice deficient in the transcription factor Ets-1.<br />
These mice die in the perinatal period with ventricular septal defects and an ectopic focus <strong>of</strong> cartilage in their<br />
outflow tract myocardium. We demonstrated that these defects are the result <strong>of</strong> altered migration and<br />
differentiation <strong>of</strong> the cardiac neural crest due to the loss <strong>of</strong> Ets-1. In this report, we describe an additional role for<br />
Ets-1 during cardiac development. We observed left ventricular systolic dysfunction in Ets-1 -/- mice as measured<br />
by a decrease in fractional shortening (45.3% vs. 22.5%, p
S5.2 Wnt signaling in the developing murine epicardium and epicardium-derived-cells<br />
(EPDCs)<br />
Carsten Rudat 1 , Julia Norden 1 , Makoto M. Taketo 2 , Andreas Kispert 1<br />
1 Institute for Molecularbiology, Hannover Medical School, Germany,<br />
2 Department <strong>of</strong> Pharmacology, Kyoto <strong>University</strong> Graduate School <strong>of</strong> Medicine, Japan.<br />
The epicardium is the outermost layer <strong>of</strong> the heart and develops from a mesothelial cluster <strong>of</strong> cells at its venous<br />
pole (the proepicardium). A subset <strong>of</strong> these cells invades the underlying subepicardium and myocardium via an<br />
epithelial-to-mesenchymal-transition (EMT). Besides this cellular contribution additional epicardium-derived<br />
paracrine signals are important for myocardial and coronary vessel development. Previous work suggested a<br />
requirement <strong>of</strong> canonical Wnt signaling in the epicardium for EMT and coronary smooth muscle cell<br />
development. Here, we reinvestigated the role <strong>of</strong> canonical Wnt signaling in the epicardium and epicardiumderived-cells<br />
during embryogenesis, using an alternative conditional genetic approach. In the background <strong>of</strong> a<br />
Tbx18cre-line showing recombination in the proepicardium and its derivatives, ß-catenin (Ctnnb1) loss- and<br />
gain-<strong>of</strong>-function alleles were analyzed. Surprisingly, mice deficient for Ctnnb1 are live born and show neither<br />
impaired coronary artery formation, nor a defective epicardium. The ability <strong>of</strong> epicardium-derived explants to<br />
differentiate into the smooth muscle lineage is maintained. Stabilization <strong>of</strong> Ctnnb1 in the epicardium resulted in<br />
lethality between embryonic days eleven and twelve. Further lineage analysis revealed the formation <strong>of</strong><br />
undifferentiated cell aggregates on the surface <strong>of</strong> the developing heart and impaired EMT.<br />
Our data contradicts previous work, by demonstrating that canonical Wnt signaling in epicardium and EPDCs is<br />
dispensable for coronary artery formation in the developing embryo. Moreover constitutive active Wnt signaling<br />
in these cells leads to early lethality, indicating only a minor physiological role <strong>of</strong> Wnt signaling in the epicardium<br />
and epicarium-derived-cells during development.<br />
46
S5.3 Determination <strong>of</strong> Cardiac Pacemaker Cell Origins Reveals a Critical Role for Wnt Signaling During<br />
their Cell Fate Specification.<br />
Michael Bressan, Gary Liu, and Takashi Mikawa<br />
<strong>University</strong> <strong>of</strong> California San Francisco, Cardiovascular research Institute<br />
Cardiac pacemaker cells have unique molecular and physiological characteristics that allow them to rapidly and<br />
autonomously generate action potentials. How and when pacemaker cell fate diversifies from adjacent<br />
myocardial cell types and are programmed to attain these specialized features, however, remains poorly<br />
understood. Here we report our fate mapping studies demonstrating that just following gastrulation pacemaker<br />
progenitors reside in the right lateral plate mesoderm posterior to the known heart fields. Our data further<br />
indicate that this pacemaker precursor region overlaps with the expression <strong>of</strong> at least one canonical Wnt,<br />
Wnt8c. This is surprising as canonical Wnts are thought to be inhibitory for myocardial specification during these<br />
stages. In order to test whether Wnt signaling is required to specifically induce pacemaker versus working<br />
myocardial fate, we injected cells expressing the soluble Wnt antagonists, Crescent, directly into the pacemaker<br />
region <strong>of</strong> gast rulating embryos. Ectopic Crescent resulted in both the expansion <strong>of</strong> the transcription factor<br />
NKX2.5 and the down-regulation <strong>of</strong> at least one pacemaker ion channel, HCN4, within the pacemaker<br />
precursors. Conversely, introduction <strong>of</strong> canonical Wnts into the heart field mesoderm was capable <strong>of</strong> inducing<br />
ectopic pacing sites and the formation <strong>of</strong> NKX2.5 negative HCN4 positive myocytes within the looping heart<br />
tube, suggesting a conversion <strong>of</strong> working myocytes into pacemaker-like cells. These data demonstrate that<br />
pacemaker progenitor specification depend on signaling cues unique from working myocardium and that Wnt<br />
signaling, at least in part, supports divergence into the pacemaker lineage. This work is supported in part by<br />
grants from the NIH-NLHBI.<br />
47
S5.4 A Tbx5-Scn5a Molecular Network Modulates Function <strong>of</strong> the Cardiac Conduction System<br />
David E Arnolds (1), Fang Liu (2), Scott Smemo (1), John P Fahrenbach (1), Gene H Kim (1), Ozanna Burnicka-<br />
Turek (1), Elizabeth M McNally (1), Marcelo M Nobrega (1), Vickas V Patel (2), and Ivan P Moskowitz (1)<br />
<strong>University</strong> <strong>of</strong> <strong>Chicago</strong>; <strong>Chicago</strong>, IL (1) and <strong>University</strong> <strong>of</strong> Pennsylvania; Philadelphia, PA (2)<br />
Cardiac conduction system (CCS) disease is common with significant morbidity and mortality. Current treatment<br />
options are limited and rational efforts to develop cell-based and regenerative therapies require knowledge <strong>of</strong><br />
the molecular networks that establish and maintain CCS function. Emerging evidence points to key CCS roles<br />
for genes with established roles in heart development. Recent genome wide association studies (GWAS) have<br />
identified numerous loci associated with human CCS function including TBX5 and SCN5A. We hypothesized<br />
that Tbx5, a critical developmental transcription factor, regulates networks required for mature CCS function.<br />
Removal <strong>of</strong> Tbx5 from the mature VCS resulted in severe VCS functional consequences, including loss <strong>of</strong> fast<br />
conduction, arrhythmias, and sudden death. Ventricular contractile function and the VCS fate map remained<br />
unchanged in VCS-specific Tbx5 knockouts. However, key mediators <strong>of</strong> fast conduction including Cx40 and<br />
Nav1.5, encoded by Scn5a, demonstrated Tbx5-dependent expression in the VCS. We identified a Tbx5responsive<br />
enhancer downstream <strong>of</strong> Scn5a sufficient to drive VCS expression in vivo, dependent on canonical<br />
T-box binding sites. Our results establish a direct molecular link between Tbx5 and Scn5a and establish a<br />
hierarchy between human GWAS loci that affects VCS function in the mature CCS, establishing a paradigm for<br />
understanding the molecular pathology <strong>of</strong> VCS disease. Ongoing studies to dissect the regulation <strong>of</strong> Scn5a<br />
expression using a Scn5a-LacZ BAC transgenic reporter system have identified two Scn5 a enhancers sufficient<br />
for establishing the native Scn5a expression pattern. Implications for the role <strong>of</strong> Scn5a and Scn10a in<br />
conduction system function, variability, and arrhythmia susceptibility will be discussed.<br />
48
S5.5 Mouse cardiac T-box targets reveal an Scn5a/10a enhancer functionally affected by genetic<br />
variation in humans<br />
Phil Barnett #,1 , Malou van den Boogaard 1 , L. Y. Elaine Wong 1 , Federico Tessadori 4 , Martijn L. Bakker 1 , Connie<br />
R. Bezzina 2 , Peter A.C. ‘t Hoen 3 , Jeroen Bakkers 4 , Vincent M. Christ<strong>of</strong>fels #,1<br />
1 Department <strong>of</strong> Anatomy, Embryology and Physiology and 2 Department <strong>of</strong> Experimental Cardiology, Heart<br />
Failure Research Center, Academic Medical Center, <strong>University</strong> <strong>of</strong> Amsterdam, The Netherlands; 3 Center for<br />
Human and Clinical Genetics and Leiden Genome Technology Center, Leiden <strong>University</strong> Medical Center,<br />
Leiden, The Netherlands; 3 Cardiac development and genetics group, Hubrecht Institute for Developmental<br />
Biology and Stem Cell Research, Utrecht, The Netherlands.<br />
# authors contributed equally and share correspondance<br />
The contraction pattern <strong>of</strong> the heart relies on the activation and conduction <strong>of</strong> the electrical impulse.<br />
Perturbations <strong>of</strong> cardiac conduction have been associated with congenital and acquired arrhythmias and cardiac<br />
arrest. The pattern <strong>of</strong> conduction depends on the regulation <strong>of</strong> heterogeneous gene expression by key<br />
transcription factors and transcriptional enhancers. Here, we assessed the genome-wide occupation <strong>of</strong><br />
conduction system regulating transcription factors Tbx3, Nkx2-5 and Gata4 and <strong>of</strong> enhancer-associated coactivator<br />
p300 in the mouse heart, uncovering cardiac enhancers throughout the genome. Many <strong>of</strong> the<br />
enhancers co-localize with ion channel genes repressed by Tbx3, including the clustered sodium channel genes<br />
Scn5a, essential for cardiac function, and Scn10a. We identify two enhancers in the Scn5a/Scn10a locus, which<br />
are regulated by Tbx3 and its family member and activator Tbx5, and are functionally conserved in humans. We<br />
provide evidence that a single-nucleotide po lymorphism in the SCN10A enhancer, associated with alterations in<br />
cardiac conduction patterns in humans, disrupts Tbx3/5 binding and reduces the cardiac activity <strong>of</strong> the enhancer<br />
in vivo. Thus, the identification <strong>of</strong> key regulatory elements for cardiac conduction helps to explain how genetic<br />
variants in non-coding regulatory DNA sequences influence the regulation <strong>of</strong> cardiac conduction and the<br />
predisposition for cardiac arrhythmias.<br />
49
S5.6 Activation <strong>of</strong> voltage-dependent anion channel 2 suppresses Ca 2+ -induced cardiac arrhythmia<br />
Hirohito Shimizu 1 , Johann Schredelseker 1 , Jie Huang 1 , Kui Lu 2 , Sarah Franklin 3 , Kevin Wang 1 , Thomas<br />
Vondriska 3 , Joshua Goldhaber 4 , Ohyun Kwon 2 , Jau-Nian Chen 1<br />
1 Department <strong>of</strong> Molecular, Cell and Developmental Biology, 2 Department <strong>of</strong> Chemistry and Biochemistry,<br />
3 Department <strong>of</strong> Anesthesiology, <strong>University</strong> <strong>of</strong> California, Los Angeles and 4 Cedars-Sinai Heart Institute, Los<br />
Angeles<br />
While a role <strong>of</strong> Ca 2+ in establishing embryonic cardiac rhythmicity has been proposed, the underlying<br />
mechanisms have yet to be fully explored. The zebrafish tremblor (tre) mutant embryo lacks functional cardiac<br />
Na + /Ca 2+ exchanger, resulting in Ca 2+ extrusion defects, abnormal Ca 2+ transients and unsynchronized cardiac<br />
contractions. We therefore use this mutant as an animal model to dissect the molecular network essential for<br />
controlling Ca 2+ homeostasis in the heart. From a chemical suppressor screen, we identified a synthetic<br />
compound named efsevin, which effectively restores synchronized cardiac contractions in tre embryos. In<br />
addition, efsevin suppresses arrhythmogenic Ca 2+ waves by accelerating the decay phase <strong>of</strong> Ca 2+ sparks in<br />
murine cardiomyocytes, demonstrating that efsevin modulates cardiac Ca 2+ handling. Through a biochemical<br />
pull-down assay, we identified a direct interaction between efsevin and the mitochondrial outer membrane<br />
protein VDAC2. Overexpression <strong>of</strong> VDAC2 restores synchronized cardiac contractions in tre embryos and<br />
knock-down <strong>of</strong> VDAC2 attenuates the rescue effect <strong>of</strong> efsevin, indicating that efsevin modulates Ca 2+ handling<br />
by enhancing the activity <strong>of</strong> VDAC2. Furthermore, overexpression <strong>of</strong> mitochondrial inner membrane Ca 2+ uptake<br />
proteins restores cardiac contractions in tre embryos. This effect is further enhanced when VDAC2 is coexpressed,<br />
and is abolished in tre/vdac2 double deficient embryos. Taken together, our data reveal an essential<br />
role for mitochondria in regulating cardiac Ca 2+ homeostasis and rhythmicity. Our findings also establish VDAC2<br />
as a critical gate for mitochondrial Ca 2+ uptake and efsevin as a potential therapeutic agent for cardiac<br />
arrhythmia.<br />
50
Platform Session 6: Early Heart Formation and Progenitors<br />
S6.1 Quantitative analysis <strong>of</strong> polarity in 3D reveals local cell coordination in the embryonic mouse heart<br />
Jean-François Le Garrec 1 , Chiara Ragni 1 , Sorin Pop 2 , Alexandre Dufour 2 , Jean-Christophe Olivo-Marin 2 ,<br />
Margaret Buckingham 1 and Sigolène Meilhac 1<br />
1 2<br />
Dept <strong>of</strong> Developmental Biology, CNRS URA 2578, Dept <strong>of</strong> Cell Biology and Infection, CNRS URA 2582,<br />
Institut Pasteur, 75015 Paris, France<br />
Several mutations in genes encoding members <strong>of</strong> the Planar Cell Polarity pathways impair heart<br />
morphogenesis. However, no asymmetric localization <strong>of</strong> such proteins has been reported in myocardial cells<br />
and it has remained unclear how the embryonic myocardium is polarized. We have now identified cell polarity<br />
markers in the mouse and aim to quantify the degree <strong>of</strong> coordination between myocardial cells in the embryonic<br />
heart. Classically, anisotropies which underlie organ morphogenesis have been quantified as a 2D problem,<br />
taking advantage <strong>of</strong> a reference axis. However, this is not applicable to the looped heart tube, which has a<br />
complex geometry. We have designed a 3D image processing framework, to map the regions in which cell<br />
polarities are significantly aligned. This novel procedure integrates multidisciplinary tools, including image<br />
segmentation, statistical analyses, axial clustering and correlation analysis. The result is a sensitive and<br />
unbiased assessment <strong>of</strong> the significant alignment <strong>of</strong> cell orientations in 3D, compared to a random axial<br />
distribution. We show that the axes <strong>of</strong> cell polarity, defined as the centrosome-nucleus axes, are frequently<br />
biased in a plane parallel to the outer surface <strong>of</strong> the heart, with a minor transmural component. This reflects the<br />
expansion <strong>of</strong> the cardiac chambers which precedes transmural thickening. Our study reveals that ventricular<br />
cells locally coordinate their axes over 100µm. This is in keeping with the growth <strong>of</strong> the myocardium, that we<br />
had shown by clonal analysis to be regionally oriented.<br />
51
S6.2 Clonally dominant cardiomyocytes direct heart morphogenesis<br />
Vikas Gupta and Kenneth D. Poss<br />
Department <strong>of</strong> Cell Biology and Howard Hughes Medical Institute, Duke <strong>University</strong> Medical Center, Durham,<br />
North Carolina 27710, USA<br />
As vertebrate embryos develop into adulthood, their organs dramatically increase in size and change tissue<br />
architecture. Here, we used a multicolor clonal analysis to define the contributions <strong>of</strong> many individual<br />
cardiomyocytes as the zebrafish heart undergoes morphogenesis from a primitive embryonic structure into its<br />
complex adult form. We find that the single cardiomyocyte-thick wall <strong>of</strong> the juvenile ventricle forms by lateral<br />
expansion <strong>of</strong> several dozen cardiomyocytes into muscle patches <strong>of</strong> variable sizes and shapes. As juveniles<br />
mature into adults, this structure becomes fully enveloped by a new lineage <strong>of</strong> cortical muscle. Adult cortical<br />
muscle originates from a small number (~8) <strong>of</strong> cardiomyocytes that display clonal dominance reminiscent <strong>of</strong><br />
stem cell populations. Cortical cardiomyocytes initially emerge from internal my<strong>of</strong>ibers that in rare events<br />
breach the juvenile ventricular wall and expand over the surface. Our study illuminates dynamic proliferative<br />
behaviors that generate adult cardiac structure, revealing clonal dominance as a key mechanism that shapes a<br />
vertebrate organ.<br />
52
S6.3 Gα 13 is required for S1pr2-mediated myocardial migration by regulating endoderm morphogenesis<br />
Ding Ye 1 , Songhai Chen 2 , Fang Lin 1 *<br />
1 Department <strong>of</strong> Anatomy and Cell Biology, 2 Department <strong>of</strong> Pharmacology, Carver College <strong>of</strong> Medicine,<br />
<strong>University</strong> <strong>of</strong> Iowa, 1-400 Bowen Science Building, 51 Newton Road, Iowa city, IA 52242-1109, USA<br />
During vertebrate development, bilateral populations <strong>of</strong> myocardial precursors migrate towards the midline to<br />
form the primitive heart tube, and this requires both their intrinsic properties and the appropriate environment,<br />
including endoderm. Disruption <strong>of</strong> myocardial migration leads to the formation <strong>of</strong> two bilaterally located hearts, a<br />
condition known as cardiac bifida. In zebrafish, signaling mediated by sphingosine-1-phosphate (S1P) and its<br />
cognate G protein-coupled receptor (S1pr2/Mil) controls myocardial migration. However, the underlying<br />
downstream mechanism remains poorly understood. Here we show that depletion <strong>of</strong> specifically the G protein<br />
is<strong>of</strong>orm Gα13, results in cardia bifida and tail blistering, defects reminiscent <strong>of</strong> those observed in embryos<br />
deficient for S1P signaling. Our genetic studies indicate that Gα13 acts downstream <strong>of</strong> S1pr2 to regulate<br />
myocardial migration through a RhoGEF/Rho-dependent pathway. Intriguingly, both cardiomyocytes and<br />
endoderm express S1pr2 and Gα13, and cardiac-specific expression <strong>of</strong> Gα13 fails to rescue cardia bifida in the<br />
context <strong>of</strong> global Gα13 inhibition. Furthermore, we found that disruption <strong>of</strong> any component <strong>of</strong> the<br />
S1pr2/Gα13/RhoGEF signaling pathway led to defects in endoderm morphogenesis that were correlated to<br />
cardia bifida, and that defects in both endoderm and myocardial migration resulting from Gα13 depletion were<br />
rescued coincidently. Overall, our findings represent the first evidence that S1pr2 controls myocardial migration<br />
through a Gα13/RhoGEF-dependent pathway, and that it does so by regulating endoderm morphogenesis.<br />
Currently, we are investigating the mechanisms by which S1pr2/Gα13 signaling controls endoderm<br />
morphogenesis.<br />
53
S6.4 A Cdc42 associated genetic network directs heart lumen formation and morphogenesis in<br />
Drosophila<br />
Georg Vogler 1 , Jiandong Liu 2 and Rolf Bodmer 1<br />
1 Development and Aging Program, Sanford-Burnham Medical Research Institute, La Jolla, CA<br />
2 Department <strong>of</strong> Biochemistry and Biophysics, <strong>University</strong> <strong>of</strong> California, San Francisco, CA.<br />
The Drosophila embryonic heart is a key model system for understanding heart specification. Our previous<br />
studies indicate that heart morphogenesis requires Slit/Robo signaling, a function conserved in vertebrates. The<br />
mechanisms by which these and other signals control heart formation are still unknown. Due to its role in<br />
membrane dynamics, we investigated the role <strong>of</strong> the small GTPase Cdc42 during Drosophila heart development<br />
and found it to be required for cardiac cell alignment and heart tube formation. Mutant or constitutively active<br />
Cdc42 in the developing heart causes improper cardioblast alignment and formation <strong>of</strong> multiple lumina,<br />
suggesting that Cdc42 is required during discrete steps <strong>of</strong> cardiogenesis. Cell polarity and filopodia dynamics<br />
are unaffected by loss <strong>of</strong> Cdc42, therefore Cdc42 might have a different role during heart morphogenesis. To<br />
understand the regulation <strong>of</strong> Cdc42 and to identify new genetic interactors, we performed a genetic screen for<br />
modifiers <strong>of</strong> Cdc42. We identified the tyrosine kinase Abelson (Abl), and the non-muscle myosin-II zipper to<br />
strongly interact with Cdc42. Abl itself shows a requirement for coordinated heart tube assembly, and Zipper<br />
exhibits a dynamic localization pattern during cardiogenesis, which depends on Cdc42 function, but is<br />
independent <strong>of</strong> Slit/Robo. Activation <strong>of</strong> the formin-like protein Diaphanous (Dia) produced defects similar to<br />
activated Cdc42, indicating that control <strong>of</strong> cell shape changes is a key regulatory step during heart<br />
morphogenesis. Our data suggest a novel mechanism <strong>of</strong> cardiac morphogenesis involving Abl, Cdc42, Dia and<br />
Zip acting in a common pathway during cardiac cell shape changes and orchestrated heart lumen formation.<br />
54
S6.5 Identification and characterization <strong>of</strong> a multipotent cardiac precursor<br />
W. Patrick Devine, Josh D. Wythe, Benoit G. Bruneau<br />
Gladstone Institute <strong>of</strong> Cardiovascular Disease, J. David Gladstone Institute<br />
San Francisco, CA 94158<br />
Construction <strong>of</strong> the vertebrate heart is a complex process involving the regulated contribution <strong>of</strong> cardiac<br />
progenitor cells in a discrete spatial and temporal order. The precision required for this process is highlighted by<br />
the frequency <strong>of</strong> congenital heart defects. Understanding the identity and regulation <strong>of</strong> these progenitors is<br />
critical to understanding the origins <strong>of</strong> congenital heart defects and has the potential to lead to novel cell-based<br />
regenerative therapies for heart disease. Previous lineage tracing studies have predicted the existence <strong>of</strong> an<br />
early, multi-potent cardiovascular progenitor, but the identity <strong>of</strong> this progenitor has remained undefined. The<br />
transcription factor Mesp1 has been postulated to mark early cardiac progenitors, however our lineage tracing<br />
results suggest that Mesp1 labels a much broader domain <strong>of</strong> cardiac and non-cardiac mesoderm than initially<br />
reported. Baf60c/Smarcd3, a subunit <strong>of</strong> the BAF chromatin-remodeling complex, is expressed specifically in the<br />
heart and somites in the early mouse embryo. We have identified an early domain <strong>of</strong> Smarcd3 expression, prior<br />
to formation <strong>of</strong> the cardiac crescent in a region <strong>of</strong> the late gastrula stage mouse embryo predicted to contain a<br />
cardiovascular progenitor population. Temporally-regulated lineage tracing <strong>of</strong> this population specifically labels<br />
both first and second heart field derived structures, including the endocardium, myocardium, and epicardium,<br />
and a population <strong>of</strong> cells within the anterior forelimb. Ongoing work will describe the precise temporal and<br />
spatial localization <strong>of</strong> Smarcd3 relative to other markers <strong>of</strong> early cardiogenesis (Tbx5, Isl1, and Nkx2-5) as well<br />
as the clonal relationship among these early Smarcd3-expressing cells.<br />
55
S6.6 Distinct origin and commitment <strong>of</strong> HCN4+ First Heart Field cells towards cardiomyogenic cell<br />
lineages<br />
Daniela Später 1,2 , Monika Abramczuk 1,2,3 , Jon Clarke 1,2 , Kristina Buac 1,2 , Makoto Sahara 1,2 , Andreas Ludwig 4 ,<br />
Kenneth R. Chien 1,2<br />
1 Cardiovascular Research Center, Massachusetts General Hospital, Boston, USA<br />
2 Harvard Stem Cell Institute, Department <strong>of</strong> Stem Cell and Regenerative Biology, Cambridge, USA<br />
3 Institute <strong>of</strong> Molecular Biotechnology <strong>of</strong> the Austrian Academy <strong>of</strong> Sciences (IMBA), Vienna, Austria<br />
4 Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Friedrich-Alexander-Universität<br />
Erlangen-Nürnberg, Germany<br />
Most <strong>of</strong> the mammalian heart is formed from two anatomically and molecularly distinct groups <strong>of</strong> cells <strong>of</strong><br />
mesodermal origin termed the first and second heart fields (FHF and SHF). Whereas the SHF gives rise to the<br />
right ventricle, parts <strong>of</strong> the atria, and proximal region <strong>of</strong> the outflow-tract, the FHF gives rise to the left ventricle<br />
and remaining parts <strong>of</strong> the atria. Multipotent progenitors <strong>of</strong> the SHF are marked preferentially by Islet-1<br />
expression, while a marker exclusive to FHF progenitors has not been identified. Here, we present Hcn4, a gene<br />
encoding the hyperpolarization-activated cyclic nucleotide-gated channel 4, as a new FHF marker expressed as<br />
early as pre-crescent stage. In-situ hybridization analysis revealed a dynamic expression <strong>of</strong> Hcn4 in the<br />
developing heart, with its strongest expression in the cardiac crescent and down-regulation by E9.5. HCN4-<br />
CreErt2 lineage tracing experiments confirm that the progeny <strong>of</strong> these cells give rise to FHF-derived structures<br />
<strong>of</strong> the heart. Surprisingly, these cells contribute primarily to cardiomyogenic cell lineages, suggesting that Hcn4+<br />
FHF precursor cells are committed cardiomyogenic progenitors from their earliest detectable stage. Single cell<br />
clonal analysis using Hcn4+ FHF cells from both mouse embryos and differentiated mouse embryonic stem cells<br />
support this finding.<br />
Our data suggests a model <strong>of</strong> cardiogenesis, where the primary purpose <strong>of</strong> the FHF is to generate cardiac<br />
muscle to support the contractile activity <strong>of</strong> the primitive heart tube, whereas the contribution <strong>of</strong> SHF-derived<br />
progenitors to the heart is more diverse, leading to cardiomyocytes, smooth muscle and endothelial cells.<br />
56
Platform Session 7: Chamber Development<br />
S7.1 irx1a Acts Downstream <strong>of</strong> nkx Genes in Maintaining Cardiac Chamber Identity in Zebrafish<br />
Sophie Colombo 1 , Vanessa George 1 , Thomas Schell 2 , Lilianna Solnica-Krezel 3 , Deborah Yelon 2,4 , and Kimara<br />
L. Targ<strong>of</strong>f 1,2<br />
1 Division <strong>of</strong> Pediatric Cardiology, Department <strong>of</strong> Pediatrics, College <strong>of</strong> Physicians & Surgeons, Columbia<br />
<strong>University</strong>, New York, NY, 10032. 2 Skirball Institute <strong>of</strong> Biomolecular Medicine, New York <strong>University</strong> School <strong>of</strong><br />
Medicine, New York, NY, 10016. 3 Department <strong>of</strong> Developmental Biology, Washington <strong>University</strong> School <strong>of</strong><br />
Medicine, St. Louis, MO, 63110. 4 Division <strong>of</strong> Biological Sciences, <strong>University</strong> <strong>of</strong> California, San Diego, La Jolla,<br />
CA, 92093.<br />
Maintenance <strong>of</strong> the features characteristic <strong>of</strong> each cardiac chamber is essential to ensure efficient and<br />
organized contractility <strong>of</strong> the heart. Despite the importance <strong>of</strong> preserving chamber-specific characteristics, the<br />
regulatory mechanisms guiding this process are yet to be uncovered. Here, we show that Nkx transcription<br />
factors are necessary to sustain ventricular attributes through repression <strong>of</strong> atrial identity. We recently isolated<br />
mutant alleles for nkx2.5 and nkx2.7, two zebrafish homologs <strong>of</strong> Nkx2-5. nkx2.5 -/- mutants exhibit a decrease in<br />
ventricular and an increase in atrial size. Loss <strong>of</strong> nkx2.7 enhances this phenotype: nkx2.5 -/- ;nkx2.7 -/- mutants<br />
display a complete absence <strong>of</strong> the ventricular chamber. The initial numbers <strong>of</strong> ventricular and atrial<br />
cardiomyocytes in mutant embryos appear normal. Yet, ventricular cardiomyocytes are lost and atrial cells are<br />
gained during chamber emergence. Additionally, analysis <strong>of</strong> chamber-specific gene expression patterns<br />
highlights a switch from a ventricular marker, vmhc, to a marker <strong>of</strong> atrial identity, amhc, suggesting<br />
transdifferentiation. Furthermore, our studies indicate that the Iroquois transcription factor Irx1a acts<br />
downstream <strong>of</strong> nkx genes. Ventricle-specific expression <strong>of</strong> irx1a is downregulated in nkx2.5 -/- and nkx2.7 -/-<br />
mutants and is absent in nkx2.5 -/- ;nkx2.7 -/- mutants. Inhibition <strong>of</strong> irx1a function leads to atrial expansion and<br />
ventricular diminution, uncovering its important role in regulating cardiac chamber proportionality. Together, our<br />
results demonstrate a pivotal role for nkx genes in maintenance <strong>of</strong> cardiac chamber identity and underscore a<br />
previously unappreciated function <strong>of</strong> irx1a. These findings have the potential to uncover etiologies <strong>of</strong> congenital<br />
heart disease in patients with NKX2-5 mutations and to direct innovations in cardiac regenerative medicine.<br />
57
S7.2 Zebrafish second heart field development relies on early specification <strong>of</strong> progenitors and nkx2.5<br />
function.<br />
Burcu Guner-Ataman 1 , Noelle Paffett-Lugassy 1,2 , Meghan S. Adams 1 , Kathleen R. Nevis 1 , Caroline E. Burns 1,2 ,<br />
C. Ge<strong>of</strong>frey Burns 1<br />
1 2<br />
Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, MA 02129; Harvard Stem<br />
Cell Institute, Cambridge, MA, 02138<br />
We recently reported that latent TGFβ binding protein 3 (ltbp3) marks the zebrafish anterior second heart field<br />
(SHF) that contributes myocardium to the distal ventricle and three cardiovascular lineages to the outflow tract<br />
(OFT). However, SHF expression <strong>of</strong> ltbp3 initiates at the arterial pole <strong>of</strong> the linear heart tube, well after higher<br />
vertebrates specify SHF progenitors in the lateral plate mesoderm (LPM). Building on a recent dye tracing study<br />
from the Kirby laboratory, we identified putative nkx2.5+ SHF progenitors in the zebrafish anterior LPM (ALPM),<br />
cranial and medial to adjacent early-differentiating cardiomyocytes (nkx2.5+, cmlc2+). Using inducible Cre/loxPmediated<br />
lineage tracing, we pulse-labeled nkx2.5+, GATA4+, or cmlc2+ cells in the ALPM and characterized<br />
their descendents using lineage specific reporters. From these analyses, we learned that pulse-labeled nkx2.5+<br />
and GATA4+ cells give rise to myocardium in the entire ventricle and three cardiovascular li neages in the OFT.<br />
Pulse-labeling <strong>of</strong> cmlc2+ cells revealed that early differentiating cardiomyocytes reside predominantly in the<br />
proximal ventricle. Taken together, these data suggest strongly that nkx2.5+, GATA4+, cmlc2- SHF progenitors<br />
are specified in the zebrafish ALPM prior to initiating expression <strong>of</strong> ltbp3 at the arterial pole. Furthermore, we<br />
tested whether nkx2.5 plays a conserved and essential role during zebrafish SHF development. Nkx2.5<br />
morphant embryos exhibited several SHF-related phenotypes including significant reductions in distal ventricular<br />
myocardium, outflow tract smooth muscle, and expression <strong>of</strong> ltbp3. Taken together, our data reveal two<br />
conserved features <strong>of</strong> zebrafish SHF development underscoring the utility <strong>of</strong> this model organism for<br />
deciphering SHF biology.<br />
58
S7.3 Notch1 mediated signaling cascades regulate cardiomyocyte polarity and ventricular wall<br />
formation.<br />
1 1 2 1 2 2 1<br />
Wenjun Zhang, Hanying Chen, Momoko Yoshimoto, Jin Zhang, Mervin C Yoder, Nadia Carlesso, Weinian<br />
Shou<br />
1<br />
Riley Heart Research Center, Herman B Wells Center for Pediatric Research, Department <strong>of</strong> Pediatrics,<br />
Indiana <strong>University</strong> School <strong>of</strong> Medicine. 2 Division <strong>of</strong> Neonatology, Herman B. Wells Center for Pediatric<br />
Research, Department <strong>of</strong> Pediatrics, Indiana <strong>University</strong> School <strong>of</strong> Medicine, Indianapolis<br />
Ventricular trabeculation and compaction are two anatomically overlapping morphogenetic events essential for<br />
normal myocardial development. Disregulation <strong>of</strong> these events can lead to Left Ventricular Noncompaction<br />
(LVNC, MIM300183). However, the underlying mechanisms and pathogenetic pathways remain elusive, largely<br />
due to the genetic heterogeneity <strong>of</strong> the patients and the lack <strong>of</strong> suitable animal models. Recently, through the<br />
use <strong>of</strong> gene pr<strong>of</strong>iling and cardiac cell-lineage restricted genetic manipulation, we analyzed a unique mouse<br />
genetic model for LVNC, Fkbp1a knockout mice. Our data demonstrated a critical contribution <strong>of</strong> over-activated<br />
Notch1 and subsequent neuregulin1-ErbB2/4 signaling in developing LVNC in mouse, and confirmed previous<br />
finding that Notch1-neuregulin1/Bmp10 signaling pathways in regulating ventricular cardiomyocyte proliferation.<br />
Remarkably, our new emerging data also suggested that the excessively activated Notch1-neuregulin1/ErbB2<br />
signaling cascade dramatically down-regulated dishevelled-associated activator <strong>of</strong> morphogenesis 1 (Daam1), a<br />
member <strong>of</strong> the Formin family and a potential effector <strong>of</strong> non-canonical Wnt planer cell polarity (PCP) signaling.<br />
Genetic ablation <strong>of</strong> Daam1 resulted in disoriented cardiomyocyte polarity with altered actin cytoskeleton and<br />
my<strong>of</strong>iber organization, and concomitantly ventricular noncompaction, which confirmed the role <strong>of</strong> Daam1 in the<br />
development <strong>of</strong> LVNC. More interestingly, we have also identified that p21-activated kinase 1 (Pak1), a Ser/Thr<br />
protein kinase known to control tyrosine kinase receptor (i.e., ErbB2/4) phosphorylation and small GTPases<br />
activities in regulating cell proliferation, cell polarity, and actin cytoskeleton organization, is a potential up-stream<br />
regulator <strong>of</strong> Daam1. Activated Pak1 (phospho-T423) was dramatically up-regulated in Fkbp1a mutant hearts<br />
with excessive activation <strong>of</strong> ErbB2/4. In addition, over-expression <strong>of</strong> constitutively active Pak1 (Pak1ca) in<br />
primary cultured neonatal cardiomyocytes down-regulated the level <strong>of</strong> Daam1. Taken together, our in vivo and<br />
in vitro data demonstrated that Notch-neuregulin-Pak1-Daam1 is an essential signaling cascade in regulating<br />
cardiomyocyte polarity and ventricular wall formation.<br />
59
S7.4 Mutations in the NOTCH pathway regulator MIB1 cause ventricular non-compaction<br />
cardiomyopathy<br />
Guillermo Luxán 1 , Jesús C. Casanova 1 , Beatriz Martínez-Poveda 1 , Donal MacGrogan 1 , Álvaro Gonzalez-Rajal 1 ,<br />
Belén Prados 1 , Gaetano D'Amato 1 , David Dobarro 1 , Carlos Torroja 2 , Fernando Martinez 2 , José Luis Izquierdo-<br />
García 3 , Leticia Fernández-Friera 3 , María Sabater-Molina 4 , Young-Y. Kong 5 , Gonzalo Pizarro 3 , Borja Ibañez 3 ,<br />
Constancio Medrano 6 , Pablo García-Pavía 7 , Juan R. Gimeno 4 , Lorenzo Montserrat 8 , Luis J. Jiménez-<br />
Borreguero 3 & José Luis de la Pompa 1<br />
1 Program <strong>of</strong> Cardiovascular Developmental Biology, Department <strong>of</strong> Cardiovascular Development and Repair,<br />
Centro Nacional de Investigaciones Cardiovasculares (CNIC), Melchor Fernández Almagro 3, 28029 Madrid,<br />
SPAIN<br />
2 Bioinformatics Unit, CNIC, Melchor Fernández Almagro 3, 28029 Madrid SPAIN<br />
3 Department <strong>of</strong> Epidemiology, Atherothrombosis and Image, CNIC, Melchor Fernández Almagro 3, 28029<br />
Madrid SPAIN<br />
4 Hospital Universitario Virgen de la Arrixaca, Ctra. Murcia-Cartagena s/n 30120 El Palmar, Murcia, SPAIN<br />
5 Department <strong>of</strong> Biological Sciences, Seoul National <strong>University</strong>, Seoul, Republic <strong>of</strong> Korea.<br />
6 Department <strong>of</strong> Cardiology, Hospital General Universitario Gregorio Marañón, Madrid, SPAIN<br />
7 Cardiomyopathy Unit. Department <strong>of</strong> Cardiology. Hospital Universitario Puerta de Hierro, Manuel de Falla 1,<br />
28222 Majadahonda SPAIN<br />
8 Instituto de Investigación Biomédica de A Coruña, Complejo Hospitalario Universitario A Coruña, As Xubias<br />
84, 15006 A Coruña, SPAIN<br />
Ventricular non-compaction (VNC) cardiomyopathy patients show prominent trabeculations mainly in the left<br />
ventricle and reduced systolic function. Clinical presentation varies from asymptomatic to heart failure. We show<br />
that germline mutations in human MIND BOMB-1 (MIB1), encoding an E3 ubiquitin ligase that promotes<br />
endocytosis <strong>of</strong> the NOTCH ligands DELTA and JAGGED, cause VNC in autosomal-dominant pedigrees, and<br />
affected individuals show reduced NOTCH1 activity. Functional studies in cells and zebrafish embryos and in<br />
silico modeling indicate that these are loss-<strong>of</strong>-function phenotypes. VNC is triggered by targeted inactivation <strong>of</strong><br />
Mib1 in mouse myocardium, and mimicked by inactivation <strong>of</strong> myocardial Jagged1 or endocardial Notch1.<br />
Myocardium-specific Mib1 mutants show reduced ventricular Notch1 activity, and an expansion <strong>of</strong> compact<br />
myocardium markers to the abnormally proliferative, immature trabeculae. These results identify NOTCH as a<br />
primary signaling pathway involved in VNC, and suggest that MIB1 mutations cause a developmental arrest in<br />
chamber myocardium, preventing trabecular maturation and compaction.<br />
60
S7.5 The early role <strong>of</strong> Tbx1 in anterior and posterior second heart field cells<br />
M. Sameer Rana 1 , Karim Mesbah 2 , Jorge N. Dominguez 3 , Niels H<strong>of</strong>schreuder 1 , Virginia E. Papaioannou 4 ,<br />
Antoon F. Moorman 1 , Vincent M. Christ<strong>of</strong>fels 1 , Robert G. Kelly 2<br />
1 Heart Failure Research Center, Department <strong>of</strong> Anatomy, Embryology and Physiology, Academic Medical<br />
Center, <strong>University</strong> <strong>of</strong> Amsterdam, Amsterdam, The Netherlands<br />
2 Developmental Biology Institute <strong>of</strong> Marseille-Luminy, Aix-Marseille <strong>University</strong>, Marseille, France<br />
3 Department <strong>of</strong> Experimental Biology, <strong>University</strong> <strong>of</strong> Jaén, Jaén, Spain<br />
4 Department <strong>of</strong> Genetics and Development, College <strong>of</strong> Physicians and Surgeons <strong>of</strong> Columbia <strong>University</strong>,<br />
Columbia <strong>University</strong> Medical Center, New York, NY, USA<br />
During cardiac development, the anterior second heart field (SHF) contributes cells to the arterial pole and the<br />
posterior SHF to the venous pole. T-box transcription factor Tbx1 is expressed in the SHF and is known to be<br />
involved in development <strong>of</strong> the arterial pole. Here, we aimed to elucidate the early role <strong>of</strong> Tbx1 in the posterior<br />
SHF and derived structures, and its relation to anterior SHF development. Using quantitative 3D reconstruction<br />
methodologies, we found that the proliferative rate and population size <strong>of</strong> SHF cells was severely affected in<br />
Tbx1-deficient mouse embryos. DiI labeling <strong>of</strong> anterior SHF cells in an Fgf10 enhancer trap transgene revealed<br />
that, in the absence <strong>of</strong> Tbx1, the anterior movement <strong>of</strong> anterior SHF cells fails from the 7 somite stage.<br />
Moreover, these anterior SHF cells contribute to the venous pole and differentiate into atrial myocardium by<br />
activating the local atrial gene program. Distal hypoplasia <strong>of</strong> the outflow tract seems to be a late consequence <strong>of</strong><br />
this defect. In addition, the distance between the outflow tract and the inflow tract was greatly reduced and the<br />
subsequent fusion <strong>of</strong> the dorsal mesocardium was consequently delayed. Our findings suggest that Tbx1 is<br />
involved in maintaining the SHF population size from embryonic day (E) 8.0 onward and that Tbx1-deficiency<br />
disrupts proliferation, differentiation and cell fate decisions within the SHF during early heart morphogenesis.<br />
Moreover, despite prepatterning into anterior and posterior subdomains, cardiac progenitor cells <strong>of</strong> the SHF<br />
seem to be naïve in the absence <strong>of</strong> Tbx1.<br />
61
S7.6 Retinol Dehydrogenase 10: Roles in Embryo Pharyngeal Patterning and a Model to Understand<br />
How Retinoid Gradients Prevent Congenital Birth Defects<br />
Muriel Rhinn a , Pascal Dollé a , Richard Finnell b , and Karen Niederreither b<br />
a IGBMC, Illkirch, France; b Dept <strong>of</strong> Nutritional Sciences, Dell Pediatric Research Institute, The <strong>University</strong> <strong>of</strong><br />
Texas, Austin.<br />
Retinoic acid (RA), an active vitamin A metabolite, is a key signaling molecule in vertebrate embryos.<br />
Morphogenetic RA gradients are thought to be set up by tissue-specific actions <strong>of</strong> retinaldehyde<br />
dehydrogenases (RALDHs) and catabolizing enzymes (CYP26s) and are important in preventing congenital<br />
malformations. The retinol to retinaldehyde conversion was believed to be achieved by several redundant<br />
enzymes; however, a random mutagenesis screen identified retinol dehydrogenase 10 (RDH10) as responsible<br />
for a homozygous lethal phenotype (Rdh10Trex mutants: Sandell et al., Genes Dev. 21, 1113-1124, 2007) with<br />
typical features <strong>of</strong> RA deficiency. We report the production and characterization <strong>of</strong> novel murine Rdh10 loss <strong>of</strong><br />
function alleles generated by gene targeting. We show that, although the Rdh10-/- mutants die at an earlier<br />
stage than Rdh10Trex mutants, their molecular patterning defects do not reflect a complete state <strong>of</strong> RA<br />
deficiency. Genetic models <strong>of</strong> RA/RALDH2 deficiency p roduces hypoplastic development <strong>of</strong> the 3rd to 6th<br />
branchial arches, a malformation disrupting cardiac outflow tract septation. In Raldh2-/- mutants this defect<br />
cannot be restored by maternally administered RA. In Rdh10-/- mutants, pharyngeal growth and patterning is<br />
restored by administering retinaldehyde to pregnant mothers. We hence obtain viable Rdh10-/- mutants, free <strong>of</strong><br />
lethal congenital malformations. This is the first demonstration <strong>of</strong> rescue <strong>of</strong> an embryonic lethal phenotype by<br />
simple maternal administration <strong>of</strong> the missing retinoid compound. These results underscore the importance <strong>of</strong><br />
maternal retinoids in preventing congenital birth defects, and lead to a revised model on the enzymatic control <strong>of</strong><br />
embryonic RA distribution.<br />
62
Platform Session 8: Valve Development<br />
S8.1 Computational modeling <strong>of</strong> endocardial cell activation during AV valve formation.<br />
Lagendijk 1 , A.K., Szabo 2 , A., Merks 2 , R. and Bakkers J. 1<br />
1 Hubrecht Institute and <strong>University</strong> Medical Center Utrecht, the Netherlands<br />
2 Netherlands Institute for Systems Biology, Amsterdam, the Netherlands<br />
The development <strong>of</strong> valve structures occurs in distinguishable phases, which are highly conserved. The first<br />
event during valve development is the induction <strong>of</strong> endocardial cushions (ECs) within the atrioventricular (AV)<br />
canal and outflow tract <strong>of</strong> the primitive heart tube. It has been well established that a BMP signal from the<br />
myocardium initiates this event by the induction <strong>of</strong> TGF-β, Has2 (hyaluronic acid synthase 2), and Notch1, as<br />
well as the transcription factors Snail1 and Twist1. The importance <strong>of</strong> regulating Has2 expression in the<br />
endocardium has been exemplified by the observation that in Has2 deficient mice or zebrafish the cardiac jelly<br />
does not expand and ECs fail to form. We recently showed that zebrafish dicer mutant embryos, lacking mature<br />
miRNAs, form excessive endocardial cushions (Lagendijk et al. Circ Res. 2011). By functional screening we<br />
found that miR-23 is both necessary and sufficient for restricting the number <strong>of</strong> activated endocardial cells in the<br />
ECs. In addition, in mouse endothelial cells, miR-23 inhibited a TGF-β–induced endothelial-to-mesenchymal<br />
transition. By in silico screening combined with in vivo testing, we identified Has2, Icat, and Tmem2 as novel<br />
direct targets <strong>of</strong> miR-23. Surprisingly, miR-23 expression is confined to the ECs where its activity is required to<br />
repress Has2. To understand the significance <strong>of</strong> the miR-23-Has2 interaction we applied computational<br />
modeling using a cellular Potts algorithm. The computational model predicts a regulatory network <strong>of</strong> both<br />
positive and negative feedback mechanisms to restrict the activation <strong>of</strong> the endocardial cells to the AV canal.<br />
63
S8.2 Tie1 is Required for Semilunar Valve Form and Function<br />
Chris Brown 1 , Xianghu Qu 1 , Kate Violette 1,2 , M-K Sewell 3 , W. David Merryman 3 , Bin Zhou, 4 and H. Scott<br />
Baldwin 1,2<br />
1 Div. <strong>of</strong> Ped. Cardiology. 2 Depts. <strong>of</strong> Cell and Dev. Biol. and 3 Biomedical Engineering, Vanderbilt <strong>University</strong>,<br />
Nashville, TN 4 Genetics, Albert Einstein College <strong>of</strong> Medicine, NY.<br />
The mechanisms regulating late gestational and early postnatal semilunar valve remodeling and maturation are<br />
poorly understood. Tie1 is a receptor tyrosine kinase with broad expression in embryonic endothelium. During<br />
semilunar valve development, Tie1 expression becomes restricted to the turbulent, arterial surfaces <strong>of</strong> the<br />
valves in the perinatal period. Previous studies in our laboratory have demonstrated that Tie1 can regulate<br />
cellular responses to blood flow and shear stress. We hypothesized that Tie1 signaling would regulate the flow<br />
dependent remodeling <strong>of</strong> the semilunar valves associated with the conversion from maternal/placental to<br />
independent neonatal circulation. To circumvent the embryonic lethality, we developed a floxed Tie1 allele and<br />
crossed it to an Nfatc1 P2-Cre line that mediates gene excision exclusively in the endocardial cushion<br />
endothelium. Excision <strong>of</strong> Tie1 resulted in aortic valve leaflets displaying hypertrophy with perturbed matrix<br />
deposition and re modeling without alteration in cell number, proliferation, or apoptosis. Differences were only<br />
detected after birth, increased with age, and were restricted to the aortic valve. RNA sequence analysis<br />
suggests that mutant aortic valves are similar to wildtype pulmonary valves in transcriptional pr<strong>of</strong>ile. The aortic<br />
valves demonstrated insufficiency and stenosis by ultrasound and atomic force microcopy documented<br />
decreased stiffness in the mutant aortic valve consistent with an increased glycosaminoglycan to collagen ratio.<br />
These data suggest that active endocardial to mesenchymal signaling, at least partially mediated by Tie1, is<br />
uniquely required for normal remodeling <strong>of</strong> the aortic but not pulmonary valve in the late gestation and post-natal<br />
animal.<br />
Suppt. by RL1HL0952551 (HSB), HL094707 (WDM), HL078881 (BZ).<br />
64
S8.3 In Vivo Reduction Of Smad2 Concomitant With Proteoglycan Accumulation Results In High<br />
Penetrance Of Bicuspid Aortic Valves<br />
1 Loren Dupuis, 2 Robert B. Hinton and 1 Christine B. Kern<br />
1 Department <strong>of</strong> Regenerative Medicine and Cell Biology, Medical <strong>University</strong> <strong>of</strong> South Carolina, Charleston, SC<br />
29464, 2 Division <strong>of</strong> Cardiology, the Heart Institute, Cincinnati Children's Hospital Medical Center, Cincinnati, OH<br />
45229<br />
Bicuspid aortic valve (BAV) is the most prevalent cardiovascular malformation resulting in two rather than three<br />
mature cusps and occurs in at least 1-2% <strong>of</strong> the population. However, only NOTCH-1 loss <strong>of</strong> function has been<br />
linked to human BAV disease, and there are limited genetically modified mouse models that display BAV. We<br />
reported that loss <strong>of</strong> proteolytic cleavage <strong>of</strong> versican, a proteoglycan abundant in cardiac outlet cushions, results<br />
in myxomatous valves in ADAMTS5 deficient mice. Recently we discovered that a failure to cleave versican was<br />
concomitant with a reduction <strong>of</strong> cell-cell condensation, phosphorylated Smad2 and fibrous ECM organization in<br />
Adamts5 -/- valves. To test the hypothesis that that versican cleavage via ADAMTS5 is required to reduce the<br />
versican-hyaluronan pericellular matrix to elicit Smad2 phosphorylation we further reduced Smad2 in Adamts5 -/-<br />
mice through intergenetic cross. The resulting Adamts5 -/- ;Smad2 +/- mice developed a more dramatic valve<br />
phenotype than Adamts5 -/-; Smad2 +/+ mice and displayed a high penetrance <strong>of</strong> BAV (6/9). Interestingly, the<br />
pulmonary valve was also bicuspid in 5/9 (4/9 displayed both bicuspid PV and BAV). All Adamts5 -/- ;Smad2 +/-<br />
semilunar valves displayed wide hinge regions at the juncture <strong>of</strong> the annulus concomitant with loss <strong>of</strong> fibrous<br />
ECM. These data suggest that the dramatic changes in proteoglycan turnover, during remodeling <strong>of</strong> the truncal<br />
cushions, elicit changes in cell behavior and signaling required for normal semilunar cusp formation. Further<br />
studies <strong>of</strong> the Adamts5 -/- ;Smad2 +/- mice may elucidate a novel etiology <strong>of</strong> BAV pathogenesis and lead to new<br />
pharmacological treatments for valve disease.<br />
65
S8.4 Filamin-A Regulates Tissue Remodeling <strong>of</strong> Developing Cardiac Valves via a Novel Serotonin<br />
Pathway<br />
Kimberly Sauls 1 , Annemarieke de Vlaming 1 , Katherine Williams 1 , Andy Wessels 1 , Robert A. Levine 2 , Susan A<br />
Slaugenhaupt 3 , Roger R. Markwald 1 , Russell A. Norris 1*<br />
1<br />
Department <strong>of</strong> Regenerative Medicine and Cell Biology, School <strong>of</strong> Medicine, Cardiovascular Developmental<br />
Biology Center, Children’s Research Institute, Medical <strong>University</strong> <strong>of</strong> South Carolina, Charleston, SC, 29425,<br />
USA<br />
2<br />
Noninvasive Cardiac Laboratory, Department <strong>of</strong> Medicine, Massachusetts General Hospital, Boston,<br />
Massachusetts, 02114, USA<br />
3<br />
Center for Human Genetic Research, Massachusetts General Hospital/Harvard Medical School, Boston,<br />
Massachusetts, 02114, USA<br />
Linkage and sequencing studies <strong>of</strong> patients with myxomatous valvular dystrophy revealed causal mutations in<br />
the Filamin-A gene. These studies defined Filamin-A as integral to valve structure and function. However, the<br />
mechanisms by which Filamin-A regulates valve cell biology are unknown. Herein we demonstrate that Filamin-<br />
A conditional KO (cKO) mice exhibit enlarged leaflets commencing during fetal valve maturation. This defect is<br />
caused by an inability for valve fibroblasts to efficiently remodel their extracellular matrix environment. Using the<br />
patient mutations as a guide, we identified a novel mechanism by which cooperative interaction between<br />
intracellular serotonin, transglutaminase-2 (TG2), and Filamin-A are required for promoting cytoskeletal-driven<br />
matrix remodeling events. Co-expression <strong>of</strong> serotonin, serotonin transporter (SERT), TG2, and Filamin-A is<br />
restricted to fetal valve development during active matrix remodeling. At this timepoint, immunoprecipitation<br />
experiments demonstrate that serotonin is covalently bound to Filamin-A in vivo and in vitro and is dependent on<br />
intact TG2 activity. Pharmacological and genetic perturbations <strong>of</strong> this interaction demonstrate serotonin<br />
transport inhibition, abrogation <strong>of</strong> TG2 activity, and/or genetic removal <strong>of</strong> Filamin-A result in loss <strong>of</strong> serotonin-<br />
Filamin-A interaction and impaired matrix remodeling. These findings illustrate a novel mechanism by which<br />
intracellular serotonin, TG2 and Filamin-A cooperatively regulate cytoskeletal-driven matrix remodeling. As<br />
such, these data provide mechanistic insight into normal processes driving cardiac valve maturation with added<br />
potential for providing insight into the developmental basis for human degenerative cardiac valve disease.<br />
66
S8.5 Epicardially-derived Fibroblasts Preferentially Contribute to the Parietal Leaflets <strong>of</strong> the<br />
Atrioventricular Valves in the Murine Heart<br />
Andy Wessels a , Maurice J. B. van den H<strong>of</strong>f b , Richard F. Adamo c , Aimee L. Phelps a Marie M. Lockhart a , Kimberly<br />
Sauls a , Laura E. Briggs a , Russell A. Norris a , Bram van Wijk b , Jose M. Perez-Pomares d , Robert W. Dettman e ,<br />
and John B. E. Burch f<br />
a<br />
Medical <strong>University</strong> <strong>of</strong> South Carolina, Charleston, South Carolina, USA.<br />
b<br />
Academic Medical Center, Amsterdam, The Netherlands.<br />
c<br />
The Children’s Hospital <strong>of</strong> Philadelphia, Philadelphia, Pennsylvania, USA.<br />
d<br />
<strong>University</strong> <strong>of</strong> Malaga, Spain.<br />
e<br />
Northwestern <strong>University</strong>, Feinberg School <strong>of</strong> Medicine, <strong>Chicago</strong>, Illinois, USA.<br />
f<br />
Fox Chase Cancer Center, Philadelphia, Pennsylvania, USA<br />
The importance <strong>of</strong> the epicardium for valvuloseptal development has been well established. To determine<br />
whether and if so how epicardially derived cells contribute to the developing valves in the murine heart we used<br />
a mWt1/IRES/GFP-Cre mouse to trace the fate <strong>of</strong> EPDCs from embryonic day (ED)10 until 4 months <strong>of</strong> age.<br />
Migration <strong>of</strong> EPDCs into the atrioventricular cushion mesenchyme starts around ED12. As development<br />
progresses, the number <strong>of</strong> EPDCs in the cushions increases significantly, specifically in the leaflets which derive<br />
from the lateral atrioventricular cushions. In these leaflets, the epicardially-derived fibroblasts eventually largely<br />
replace the endocardially-derived cells. Importantly, the contribution <strong>of</strong> EPDCs to the leaflets derived from the<br />
major AV cushions is very limited. The differential contribution <strong>of</strong> EPDCs to the respective leaflets <strong>of</strong> the<br />
atrioventricular valves provides a new paradigm in valve development and could lead to new insights into the<br />
pathogenesis <strong>of</strong> abnormalities that preferentially affect individual components <strong>of</strong> this region <strong>of</strong> the heart. The<br />
notion that there is a significant difference in the contribution <strong>of</strong> epicardially and endocardially derived cells to<br />
the individual leaflets <strong>of</strong> the atrioventricular valves has also important pragmatic consequences for the use <strong>of</strong><br />
endocardial and epicardial cre-mouse models in heart development.<br />
67
S8.6 Endothelial nitric oxide signaling regulates Notch1 in aortic valve development and disease<br />
Kevin Bosse PhD, Chetan P. Hans PhD, Ning Zhao MS, Sara N. Koenig BA, Nianyuan Huang BS, Anuradha<br />
Guggilam PhD, Ge Tao PhD, Pamela A. Lucchesi PhD, Joy Lincoln PhD, Brenda Lilly PhD and Vidu Garg MD<br />
The Research Institute at Nationwide Children's Hospital<br />
Aortic valve calcification is the third leading cause <strong>of</strong> heart disease in adults and is frequently associated with<br />
bicuspid aortic valve, the most common type <strong>of</strong> cardiac malformation. NOTCH1 and the nitric oxide (NO)<br />
signaling pathway have been implicated in aortic valve development and calcification. Using an established<br />
porcine aortic valve interstitial cell (PAVIC) culture system known to spontaneously calcify, we demonstrate that<br />
gain or loss <strong>of</strong> NO, secreted by endothelial cells, prevents or accelerates calcification <strong>of</strong> PAVICs, respectively.<br />
Overexpression <strong>of</strong> Notch1 prevented the calcification that occurred with inhibition <strong>of</strong> NO, demonstrating the<br />
effects <strong>of</strong> NO on calcification are mediated by Notch1. Furthermore, endothelial cells or addition <strong>of</strong> NO<br />
regulates the nuclear localization <strong>of</strong> Notch1 and affects the expression <strong>of</strong> Hey1, a Notch1 target gene in<br />
PAVICs. To determine if Notch1 and endothelial nitric oxide (eNOS) genetically interact in vivo, we generated<br />
eNOS -/- ;Notch1 +/- compound mutant mice. Embryonic examination revealed cardiac abnormalities in a subset <strong>of</strong><br />
eNOS -/- ;Notch1 +/- mice, which suffered ~70% lethality by postnatal day 10. Surviving compound mutant mice at<br />
8 weeks <strong>of</strong> age demonstrated severely malformed aortic valves. eNOS -/- ;Notch1 +/- mice maintained on high fat<br />
diet for 12 weeks displayed aortic regurgitation and stenosis, thickened aortic cusps and development <strong>of</strong> calcific<br />
nodules on the aortic valve leaflets as compared to age-matched eNOS -/- mice. Overall, these data<br />
demonstrate a novel molecular pathway by which endothelial nitric oxide regulates Notch1 signaling in a manner<br />
that is critical for aortic valve development and disease.<br />
68
Session 1: Cardiac Regeneration<br />
Moderated Poster Discussions<br />
2.7 Cardiomyocyte proliferation contributes to post-natal heart growth in humans<br />
Kevin Bersell 1 , Mariya Mollova 1 , Stuart Walsh 1 , Jainy Savla 1 , Tanmoy DasLala 1 , Shin-Young Park 1 , Leslie<br />
Silberstein 1 , Cris dosRemedios 2 , Dionne Graham 1 , Steven Colan 1 and Bernhard Kühn 1<br />
1 Children's Hospital Boston, 2 <strong>University</strong> <strong>of</strong> Sydney<br />
Cardiomyocyte proliferation is an active cellular mechanism during myocardial growth and regeneration in<br />
zebrafish, newt, and neonatal mice. We hypothesized that cardiomyocyte proliferation may also contribute to the<br />
growth <strong>of</strong> the human myocardium between birth and adulthood. To test this hypothesis, we analyzed the<br />
cellular growth mechanisms in a set <strong>of</strong> 20 human hearts (age 3 weeks – 20 years) that were procured for<br />
transplantation and free from myocardial diseases. Measurements <strong>of</strong> mitosis, made by automated<br />
quantification <strong>of</strong> phosphorylated histone-3-positive cardiomyocytes, showed that during the first year <strong>of</strong> life the<br />
mean percentage <strong>of</strong> cardiomyocytes undergoing mitosis was 0.05%, declining to 0.01% at 20 years <strong>of</strong> age.<br />
Cardiomyocyte cytokinesis, visualized by an antibody against MKLP-1 (a component <strong>of</strong> the centralspindlin<br />
complex), was detectable up to 20 years <strong>of</strong> age, but not later in life. The number <strong>of</strong> cardiomyocytes, quantified<br />
with stereology, increased four-fold between birth and 20 years. The mean cardiomyocyte volume increased<br />
concomitantly 12-fold. Relating these mechanistic data to left ventricular mass (determined by<br />
echocardiography) showed that cardiomyocyte proliferation contributes 37% to physiologic myocardial growth<br />
between birth and 20 years. These findings suggest that altered cardiomyocyte proliferation may be involved in<br />
abnormal myocardial growth in congenital heart disease. Our findings also suggest that myocardial regeneration<br />
may be present or stimulated in young humans since the underlying mechanism, cardiomyocyte proliferation, is<br />
active under the age <strong>of</strong> 20 years.<br />
2.8 Microarray and Ultrastructure Analyses <strong>of</strong> a Regenerative Myocardium<br />
Pooja Pardhanani 1 , Jeremy Barth 2 , Heather J. Evans-Anderson 1<br />
1 Winthrop <strong>University</strong>, Rock Hill, SC 29733<br />
2 Medical <strong>University</strong> <strong>of</strong> South Carolina, Charleston, SC 29425<br />
Ciona intestinalis is an invertebrate animal model system that is well characterized and has many advantages<br />
for the study <strong>of</strong> cardiovascular biology. A striking difference between most vertebrates and Ciona is that the<br />
Ciona myocardium is capable <strong>of</strong> regenerating cardiac myocytes throughout its lifespan, which makes the<br />
regulatory mechanisms <strong>of</strong> cardiac myocyte proliferation in Ciona intriguing. In order to identify important<br />
regeneration factors in Ciona, microarray analysis was conducted on RNA from adult Ciona hearts with normal<br />
or damaged myocardium using custom Affymetrix GeneChips. Hearts were injured via ligation or cryoinjury to<br />
stimulate regeneration. After a 24 or 48 hour recovery period, total RNA was isolated from damaged and control<br />
hearts. Initial results indicate significant changes in gene expression in hearts damaged by ligation in<br />
comparison to cryoinjured or control hearts. Ligation injury shows differential expression <strong>of</strong> 223 genes as<br />
compared to control (fold change >2, p
could provide the basis for a therapeutic approach for the treatment <strong>of</strong> cardiac dysfunction following myocardial<br />
infarction. In contrast to the adult mammalian heart, the heart <strong>of</strong> one-day old mice maintains the capacity to<br />
completely regenerate after partial resection, by proliferation <strong>of</strong> mature CMs. The main objective <strong>of</strong> our work is<br />
to decipher the structural and molecular differences between CMs <strong>of</strong> adult versus neonatal hearts, in attempts to<br />
improve the regenerative capacity <strong>of</strong> the adult mammalian heart. For that, we use multidisciplinary approaches<br />
enabling to study the molecular and cellular mechanisms that govern CM proliferation, combining various<br />
structural and molecular cell-biology methodologies, using both cell lines and primary cell cultures. Specifically,<br />
we characterized CM populations derived from 1d and 7d old mice and determined the expression pr<strong>of</strong>ile <strong>of</strong><br />
their cardiogenic genes, as well as their proliferation, differentiation and de-differentiation capacity in vitro. In<br />
addition to this characterization, we study how the composition, geometry and forces <strong>of</strong> the extracellular<br />
environment affect the specific features <strong>of</strong> CMs. Using high-throughput live-cell imaging platforms, we test<br />
various signalling pathways for their ability to prime the proliferation, differentiation and de-differentiation <strong>of</strong><br />
CMs. Preliminary results and future directions <strong>of</strong> the combined strategy <strong>of</strong> studying both the traits <strong>of</strong> CMs<br />
together with those <strong>of</strong> the microenvironment and the possible crosstalk between will be described.<br />
2.10 Tissue specific translational pr<strong>of</strong>iling during zebrafish heart regeneration<br />
Yi Fang, Vikas Gupta, Ravi Karra, Taylor Wahlig, Jennifer Holdway, Jinhu Wang, and Kenneth D. Poss<br />
Department <strong>of</strong> Cell Biology and Howard Hughes Medical Institutes, Duke <strong>University</strong> Medical Center, Durham,<br />
North Carolina, USA<br />
Zebrafish have a robust ability to replace lost cardiomyoyctes, and thus provide a unique model to dissect heart<br />
regeneration. Genetic lineage-tracing has revealed that regeneration occurs by activating proliferation <strong>of</strong> preexisting<br />
cardiomyocytes at the injury site. In particular, cardiomyocytes that activate expression <strong>of</strong> the<br />
transcription factor Gata4 are the major contributors <strong>of</strong> new cardiac muscle. Yet, the molecular mechanisms by<br />
which injury activates cardiomyocyte proliferation remain elusive. Here, we have applied the recently developed<br />
technology, translational affinity purification (TRAP), to obtain genome-wide pr<strong>of</strong>iles <strong>of</strong> translated mRNAs from<br />
different cardiac cell types during regeneration. We created transgenic lines to pr<strong>of</strong>ile cardiomyocytes, gata4positive<br />
cardiomyocytes, epicardium, and endocardium, at various times after two different models <strong>of</strong> cardiac<br />
injury. Through translational pr<strong>of</strong>iling <strong>of</strong> cardiomyocytes, we have begun to identify new injury responses by<br />
cardiomyocytes that are critical for heart regeneration in zebrafish.<br />
Session 2: Human Genetics & Disease Models<br />
1.7 Novel role <strong>of</strong> altered cardiac function in the progression <strong>of</strong> heart defects in Fetal Alcohol Syndrome<br />
(FAS)<br />
Ganga Karunamuni 1 , Shi Gu 2 , Lindsy Peterson 2 , Zhao Liu 2 , Yong Qiu Doughman 1 , Kersti Linask 3 , Michael<br />
Jenkins 2 , Andrew Rollins 2 , Michiko Watanabe 1<br />
1 2 3<br />
Pediatrics and Biomedical Engineering, Case Western Reserve <strong>University</strong>, Cleveland, OH 44106, Pediatrics,<br />
<strong>University</strong> <strong>of</strong> South Florida, St. Petersburg, FL 33701<br />
Over 500,000 American women per year report drinking alcohol during pregnancy, with 1 in 5 who also binge<br />
drink. Even low levels <strong>of</strong> prenatal alcohol/ethanol exposure can produce birth defects in humans.<br />
Epidemiological studies suggest that 54% <strong>of</strong> live-born children with Fetal Alcohol Syndrome (FAS) present with<br />
cardiac anomalies. While the mechanisms <strong>of</strong> ethanol exposure have been studied extensively, most studies fail<br />
to consider the role <strong>of</strong> altered cardiac function in producing congenital heart defects. It is already known that<br />
changes in hemodynamics can pr<strong>of</strong>oundly affect cardiac development. We hypothesized that ethanol exposure<br />
creates early hemodynamic anomalies which contribute significantly to subsequent cardiac structural and<br />
functional defects. We employed optical coherence tomography (OCT), which is a non-destructive imaging<br />
modality capable <strong>of</strong> real-time, micrometer-scale resolution imaging. OCT allowed us to accurately map changes<br />
in hemodynamic forces (e.g. shear stress) and the resultant structural abnormalities in the live embryo at very<br />
early stages, when the trajectory to heart defects can begin. In our studies, avian embryos exposed to ethanol<br />
during gastrulation exhibited alterations in overall embryo body flexure, blood flow, shear stress and cardiac<br />
cushion development during heart looping stages. A combination <strong>of</strong> myo-inositol (MI) and folate (FA) given at<br />
gastrulation stages are known to prevent ethanol-induced cardiac abnormalities in mouse/avian models. In<br />
future studies, we will test whether FA/MI supplementation rescues these defects through normalization <strong>of</strong><br />
cardiac function. Our contributions could be a first step in implementing new therapeutic strategies based on<br />
FA/MI prevention <strong>of</strong> birth defects. Research Support: HL083048, HL095717.<br />
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1.8 Noonan syndrome associated RAF1 mutant evokes hypertrophic cardiomyopathy features in human<br />
cardiomyocytes in vitro.<br />
Malte Tiburcy 1 , Nicole Dubois 2 , Aleksander Hinek 3 , Seema Mital 3 , Darrell Kotton 4 , Wolfram Zimmermann 1 ,<br />
Gordon Keller 2 , Benjamin Neel 2 , Toshiyuki Araki 2<br />
1 <strong>University</strong> <strong>of</strong> Gottingen, 2 <strong>University</strong> Health Network, 3 Hospital for Sick Children, Boston <strong>University</strong> 4<br />
Noonan syndrome (NS) and its related disorders, which are now called “RASopathies”, are caused by aberrant<br />
activation <strong>of</strong> RAS/ERK pathway. Most RASopathies feature proportional short stature, facial dysmorphia,<br />
cognitive impairment and cardiac defects. The cardiac manifestations in RASopathies vary widely, but<br />
hypertrophic cardiomyopathy (HCM) is found in virtually all NS cases caused by RAF1 allele that encode a<br />
hyperactive kinase mutant. HCM also is common in LEOPARD syndrome and Costello syndrome. We reported<br />
previously that a mouse model <strong>of</strong> NS caused by a kinase-activated Raf1 mutant recapitulates major features <strong>of</strong><br />
NS, including HCM. Importantly, these features were normalized by post-natal treatment <strong>of</strong> MEK inhibitor. To<br />
extend these mouse studies to pre-clinical human models to better identify detail molecular basis and signaling<br />
as well as to aid in the development <strong>of</strong> new therapies for RASopathies, we have generated human induced<br />
pluripotent stem cells (hiPSCs) from fibroblasts <strong>of</strong> multiple RASopathy patients. We found that a kinaseactivating<br />
RAF1 mutant causes increased cell size <strong>of</strong> cardiomyocytes differentiated from hiPSCs compared with<br />
normal hiPSCs. We also found increased calcium sensitivity and lower response to b adrenergic stimulation<br />
when we cultured differentiated cardiomyocytes as engineered heart tissue, which allows us to apply<br />
mechanical forces on these cardiomyocytes. Our data show that NS associated RAF1 mutant can cause HCM<br />
phenotypes in vitro and provide a potential pre-clinical system for testing new therapies.<br />
1.9 Engineering new mouse models to map dosage-sensitive genes in Down syndrome congenital heart<br />
defects<br />
Eva Lana-Elola 1 , Sheona Watson-Scales 1 , Amy Slender 1 , Louisa Dunlevy 1 , Mike Bennett 1 , Timothy Mohun 1 ,<br />
Elizabeth M. C. Fisher 2 , Victor L. J. Tybulewicz 1<br />
1<br />
MRC, National Institute for Medical Research, London, UK<br />
2<br />
UCL Institute <strong>of</strong> Neurology, London, UK<br />
Trisomy <strong>of</strong> human chromosome 21 (Hsa21) occurs in ~1 in 750 live births and the resulting gene dosage<br />
imbalance causes Down syndrome (DS). One <strong>of</strong> the most important medical aspects are the congenital heart<br />
defects (CHD), which affect around half <strong>of</strong> all individuals with DS. The defects observed in DS range from<br />
simple chamber septation to complex CHD involving multiple aspects <strong>of</strong> the heart anatomy. The most common<br />
CHD in DS affect the atrio-ventricular (AV) junction. Using high-resolution episcopic microscopy (HREM) in<br />
mouse embryonic hearts, we have studied a ‘transchromosomic’ (Tc1) mouse model <strong>of</strong> DS, which carries<br />
Hsa21 as a freely segregating chromosome. The Tc1 mouse closely resembles human DS CHD, replicating<br />
many <strong>of</strong> the features <strong>of</strong> the AV septal defects found in DS (Dunlevy, 2010). To unravel the mechanisms <strong>of</strong><br />
defective heart formation in the Tc1 mouse embryo, we are using lineage markers to study the tissues involved<br />
in development <strong>of</strong> the AV junction. In order to identify dosage-sensitive genes that may underlie DS CHD and<br />
other complex biological effects <strong>of</strong> trisomy, we are generating a high-resolution mapping panel <strong>of</strong> mouse strains<br />
with partial trisomies and monosomies for regions <strong>of</strong> mouse chromosomes orthologous to Hsa21. Using<br />
systematic HREM analysis, we are examining partial trisomic strains to identify regions that are sufficient to<br />
induce the CHD phenotype. Conversely, by crossing Tc1 mice to partial monosomic strains and assaying the<br />
effect <strong>of</strong> each interval on the cardiac phenotype, we are testing individual genes for their potential role in DS<br />
CHD.<br />
1.10 Recovery <strong>of</strong> ENU induced mutations causing congenital heart disease using next-gen sequencing<br />
You Li, Sarosh Fatakia, Ashok Srinivasan, Histao Yagi, Rama Damerla, Bishwanath Chatterjee, Cecilia W. Lo<br />
Dept <strong>of</strong> Developmental Biology, <strong>University</strong> <strong>of</strong> Pittsburgh School <strong>of</strong> Medicine, Pittsburgh, PA<br />
Forward genetic screens provide a powerful non-gene biased approach to elucidate the genetic basis for<br />
congenital heart disease (CHD). In the past this has been hampered by difficulties in mutation recovery. In our<br />
current large-scale recessive mouse ENU mutagenesis screen, we are pursuing the use <strong>of</strong> whole<br />
genome/whole exome sequencing for mutation recovery. To evaluate the efficacy <strong>of</strong> our mutation recovery<br />
pipeline, the mutant Destro exhibiting complex CHD with heterotaxy was analyzed by both whole exome and<br />
whole genome sequencing. A multi-step bioinformatics filtering strategy was developed for mutation recovery.<br />
First, we removed sequence variants present in dbSNP129 or our in-house mouse databases. Second, coding<br />
71
variants likely to be homozygous based on sequence coverage, unique reads, and strandedness were<br />
prioritized as potential candidates. Third, mutations are further rank ordered based on cross-species<br />
conservation. Finally, the causal mutation is identified by genotyping multiple mutants with the same phenotype.<br />
Whole-genome sequencing <strong>of</strong> Destro with 8x coverage together with our filtering strategy reduced 178,848<br />
variants to just 6, with genotyping analysis identifying Bicc1 c.606+2T>C as the disease-causing mutation. This<br />
same mutation was also recovered by whole-exome sequencing with ~50x target-coverage. In another mutant<br />
line 370 with coarctation and diaphragmatic hernia, exome sequencing analysis yielded a single mutation in<br />
Lox1, which was confirmed to be the disease causing mutation. Over 10 mutations have been recovered thus<br />
far. These results suggest our whole exome-sequencing pipeline will provide a cost-effective strategy for<br />
meeting our goal <strong>of</strong> pursuing a saturation level mutagenesis screen to recover mutations causing CHD.<br />
Supported by funding from U01-HL098180<br />
Session 3: Transcriptional Regulation<br />
3.7 Mutual Regulation <strong>of</strong> Nkx2.5 and Fibulin-1 in the SHF Regulatory Network<br />
Anthony J. Horton 1 , Marion A. Cooley 2 , Waleed O. Twall 2 , Victor M. Fresco 2 , Boding Zhang 1 , Christopher D.<br />
Clark 1 , W. Scott Argraves 2 and Kyu-Ho Lee 1*<br />
1 Children’s Hospital, Division <strong>of</strong> Pediatric Cardiology and 2 Department <strong>of</strong> Regenerative Medicine and Cell<br />
Biology, Medical <strong>University</strong> <strong>of</strong> South Carolina, Charleston, SC 29425<br />
Approximately 12,000 children are born yearly with malformations <strong>of</strong> the aorta and/or pulmonary artery resulting<br />
from abnormal outflow tract (OFT) development. The cardiac OFT arise from progenitor cells <strong>of</strong> the second<br />
heart field (SHF) and animal studies have shown that this process is subject to the influence <strong>of</strong> several<br />
transcription factor, growth factor and extracellular matrix (ECM) genes. It is therefore likely that the<br />
combinatorial effects <strong>of</strong> multiple gene mutations underlie genetically complex OFT congenital heart defects<br />
(CHD) entities in humans. As part <strong>of</strong> an effort to determine functional linkages between known OFT<br />
developmental regulators, we have found evidence for mutual regulation <strong>of</strong> the cardiac transcription factor<br />
Nkx2.5 and the ECM protein fibulin-1 (Fbln1), both <strong>of</strong> which are required for normal SHF and OFT development<br />
in mice. Specifically we have found that Fbln1 expression levels are decreased in SHF regions in Nkx2.5 null<br />
mice, and that Nkx2.5 appears to regulate Fbln1 expression in the SHF via binding to an evolutionarily<br />
conserved NKE binding site in promoter proximal regions. Conversely, Fbln1 expression reciprocally regulates<br />
Nkx2.5, as Nkx2.5 expression is decreased in SHF regions <strong>of</strong> Fbln1 null mice in association with increased<br />
expression <strong>of</strong> a potential direct repressor <strong>of</strong> Nkx2.5, the growth factor activated transcription factor Egr1. These<br />
findings support the hypothesis that Nkx2.5 and Fbln1 are key components <strong>of</strong> a growth factor-mediated<br />
homeostatic mechanism regulating SHF proliferation and OFT development.<br />
3.8 FOG-2 Mediated Recruitment <strong>of</strong> the NuRD Complex Regulates Cardiomyocyte Proliferation during<br />
Heart Development<br />
Audrey Jerde, Zhiguang Gao, Spencer Martens, Judy U. Early, Eric C. Svensson<br />
Department <strong>of</strong> Medicine, The <strong>University</strong> <strong>of</strong> <strong>Chicago</strong>, <strong>Chicago</strong>, IL, 60637<br />
FOG-2 is a multi-zinc finger protein that binds GATA4 and modulates GATA4-mediated transcriptional control <strong>of</strong><br />
target genes during heart development. Our previous work has demonstrated that the Nucleosome Remodeling<br />
and Deacetylase (NuRD) complex physically interacts with FOG-2 and is necessary for FOG-2 mediated<br />
repression <strong>of</strong> GATA4 activity in vitro. In this report, we describe the generation and characterization <strong>of</strong> mice<br />
homozygous for a mutation that disrupts FOG-2/NuRD complex interaction (FOG-2 R3K5A ). These mice exhibit a<br />
perinatal lethality and have multiple cardiac malformations, including a ventricular septal defect and a thin<br />
ventricular myocardium. To investigate the mechanism underlying the thin ventricular walls, we measured the<br />
rate <strong>of</strong> cardiomyocyte apoptosis and proliferation in wild-type and FOG-2 R3K5A developing hearts. We found no<br />
significant difference in the rate <strong>of</strong> cardiomyocyte apoptosis between wild-type and FOG-2 R3K5A mice. However,<br />
cardiomyocyte proliferation was reduced by 29.8% in FOG-2 R3K5A mice. Interestingly, we found by gene<br />
expression analysis that the cell cycle inhibitor p21 is up-regulated 1.7-fold in FOG-2 R3K5A hearts. Further, we<br />
demonstrate that FOG-2 can directly repress the activity <strong>of</strong> the p21 gene promoter using an in vitro transient<br />
transfection assay. In addition, chromatin immunoprecipitation (ChIP) reveals that the NuRD complex is bound<br />
to the p21 promoter in wild-type embryonic day 14.5 hearts but is diminished at this promoter in mutant hearts.<br />
Taken together, our results suggest the FOG-2/NuRD interaction is required for cardiomyocyte proliferation by<br />
specifically down-regulating expression <strong>of</strong> the cell cycle inhibitor p21 during heart development.<br />
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3.9 A strict lineage boundary between the first and second heart fields is defined by the contribution <strong>of</strong><br />
the Tbx5 lineage.<br />
Joshua D. Wythe, W. Patrick Devine, Kazuko Koshiba-Takeuchi, Kyonori Togi, Benoit G. Bruneau<br />
Gladstone Institute <strong>of</strong> Cardiovascular Disease, <strong>University</strong> <strong>of</strong> California, San Francisco. 1650 Owens Street, San<br />
Francisco, CA 94158, USA.<br />
The theory that the heart arises from two distinct heart fields suggests important clues as to how the heart is<br />
patterned. Determining if contributions <strong>of</strong> distinct first heart field (FHF) and second heart field (SHF) cell<br />
populations to the fully formed heart exist is central to understanding heart development and the etiology <strong>of</strong><br />
congenital heart defects (CHDs). Haploinsufficiency <strong>of</strong> the transcription factor TBX5 causes Holt-Oram<br />
Syndrome, which includes CHDs. Tbx5 expression outlines a region <strong>of</strong> the heart that is presumed to correspond<br />
to the FHF, and Tbx5 is required in the posterior half <strong>of</strong> the heart. However, it is not known what structures<br />
within the heart the Tbx5 lineage gives rise to, or how these relate to the importance <strong>of</strong> Tbx5 in CHDs. Using an<br />
inducible Cre recombinase inserted at the Tbx5 locus, and various SHF genetic markers, we have mapped the<br />
Tbx5-expressing lineage and its intersection with the SHF, to discern field allocation in cardiac morphogenesis.<br />
We fi nd that from the earliest stages <strong>of</strong> Tbx5 expression, prior to formation <strong>of</strong> the linear heart tube, the Tbx5<br />
lineage contributes to the posterior segments <strong>of</strong> the heart, ending at a sharp boundary at the interventricular<br />
septum, with little overlap with the SHF. We propose that the earliest Tbx5 expressing cells define the FHF<br />
lineage, and that a strict lineage boundary exists between the FHF and SHF prior to morphogenic distinctions<br />
between their descendents. Understanding the contribution <strong>of</strong> Tbx5 to the FHF will contribute to our knowledge<br />
<strong>of</strong> heart development and CHDs.<br />
3.10 Tbx5-Hedgehog pathway is required in second heart field cardiac progenitors for atrial septation<br />
Linglin Xie 1, 2 , Andrew D. H<strong>of</strong>fmann 1 , Ozanna Burnicka-Turek 1 , Joshua M. Friedland-Little 1 , Ke Zhang 3 , Ivan P.<br />
Moskowitz 1<br />
1 Departments <strong>of</strong> Pediatrics and Pathology, The <strong>University</strong> <strong>of</strong> <strong>Chicago</strong>, <strong>Chicago</strong>, IL, 60637, USA; 2 Department <strong>of</strong><br />
Biochemistry and Molecular Biology and 3 Department <strong>of</strong> Pathology, The <strong>University</strong> <strong>of</strong> North Dakota, Grand<br />
Forks, ND, 58202, USA<br />
The developmental mechanisms underlying human Congenital Heart Disease (CHD) are poorly established.<br />
Atrial Septal Defects (ASDs), a common form <strong>of</strong> CHD, can result from haploinsufficiency <strong>of</strong> cardiogenic<br />
transcription factors including Tbx5. We demonstrate that Tbx5 is required outside the heart in second heart<br />
field (SHF) cardiac progenitors for atrial septation in mice. Conditional Tbx5 haploinsufficiency in the SHF, but<br />
not in the myocardium or endocardium, caused ASDs. Tbx5 SHF knockout embryos lacked atrial septum<br />
progenitors. We identified SHF Tbx5- responsive cell cycle progression genes, including cdk6 as a direct target,<br />
and Tbx5 mutant SHF progenitors demonstrated a mitotic defect. Genetic and molecular evidence including the<br />
rescue <strong>of</strong> atrial septation in Tbx5 mutant embryos by constitutive Hh-signaling placed Tbx5 upstream or parallel<br />
to Hh signaling in cardiac progenitors. We identified Gas1, a Hh-pathway member upstream <strong>of</strong> Smo, as a direct<br />
target <strong>of</strong> Tbx5. We also found that Osr1, required for atrial septaion in mice, is expressed in a Tbx5-dependent<br />
manner. We further identified Osr1 as a direct target <strong>of</strong> Tbx5 required for atrial septation. These results<br />
describe a SHF Tbx5-Hh-signaling network and independent Tbx5-Osr1 pathway required for atrial septation.<br />
Ongoing work is aimed at identifying molecular links between these pathways in atrial septation. A paradigm<br />
defining molecular requirements downstream <strong>of</strong> Tbx5 in cardiac progenitors as organizers <strong>of</strong> cardiac septum<br />
morphogenesis has implications for the ontogeny <strong>of</strong> CHD.<br />
Session 4: Coronary Artery Development<br />
5.7 The BMP regulator BMPER is necessary for normal coronary artery formation<br />
Laura Dyer and Cam Patterson<br />
McAllister Heart Institute, <strong>University</strong> <strong>of</strong> North Carolina at Chapel Hill<br />
Connection <strong>of</strong> the coronary vasculature to the aorta is one <strong>of</strong> the last essential steps <strong>of</strong> cardiac development.<br />
However, little is known about the signaling events that promote normal coronary artery formation. The bone<br />
morphogenetic protein (BMP) signaling pathway regulates multiple aspects <strong>of</strong> endothelial cell biology but has<br />
not been specifically implicated in coronary vascular development. BMP signaling is tightly regulated by<br />
numerous factors, including BMP-binding endothelial cell precursor-derived regulator (BMPER), which can both<br />
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promote and repress BMP signaling activity. Analysis <strong>of</strong> the BMPER-/- mouse indicated that this deficiency<br />
leads to embryonic death, and in the embryonic heart, BMPER expression is limited to the endothelial cells and<br />
the endothelial-derived cushions, suggesting that coronary vascular defects may be present. Histological<br />
analysis <strong>of</strong> BMPER-/- embryos at embryonic day 16.5 revealed that the coronary arteries were either atretic or<br />
connected distal to the semilunar valves. However, analysis <strong>of</strong> earlier embryonic stages showed that the<br />
coronary plexus began differentiating normally and that apoptosis and cell proliferation were unaffected in<br />
BMPER-/- embryos. In vitro tubulogenesis assays showed that isolated BMPER-/- endothelial cells had impaired<br />
tube formation compared to wild-type endothelial cells. Together, these results indicate that BMPER-regulated<br />
BMP signaling is critical for the migration <strong>of</strong> coronary endothelial cells and normal coronary artery development.<br />
5.8 Epicardial chemokine signaling influences ventricular wall proliferation and coronary<br />
vasculogenesis<br />
Susana Cavallero, Hua Shen, Peng Li, Henry M. Sucov.<br />
Broad Center for Regenerative Medicine and Stem Cell Research, Keck School <strong>of</strong> Medicine, <strong>University</strong> <strong>of</strong><br />
Southern California, Los Angeles, CA.<br />
During heart development, the epicardium secretes soluble factors that direct the morphological organization <strong>of</strong><br />
the underlying myocardial wall. Our previous study established that insulin-like growth factor 2 (IGF2) is the<br />
primary mitogen expressed by the epicardium that controls embryonic ventricular cardiomyocyte proliferation.<br />
We found that CXCL12 (stromal derived factor-1) is an additional factor expressed by epicardial cells in vitro<br />
and by the epicardium in vivo. Cultured primary cardiomyocytes treated with epicardial conditioned media or in<br />
coculture with epicardial cells show a proliferative response that is partially blocked by AG1024 (an IGFR<br />
inhibitor) or AMD3100 (a CXCR4 inhibitor), and almost completely abolished by both compounds together. To<br />
date, a functional role for CXCL12 signaling in heart development has not been examined. Our results indicate<br />
that both global Cxcl12 null and cardiac-specific (Nkx2.5Cre) Cxcr4 null embryos show a moderate deficiency in<br />
compact zone proliferation. Embryos with combined loss <strong>of</strong> IGF2 and CXCL12 signaling exhibit pronounced<br />
edema, suggesting that epicardial IGF2 and CXCL12 together promote mitogenic signaling needed for<br />
cardiomyocyte proliferation and compact zone expansion. Because CXCL12 is a chemokine that has known<br />
effects on vascular cells, we also examined coronary vessel formation in embryos with disrupted CXCL12<br />
signaling. We observed expanded superficial vessels and a reduced number <strong>of</strong> intramyocardial arteries. Our<br />
results indicate that the epicardium is a source <strong>of</strong> secreted CXCL12 that participates with the major mitogen<br />
IGF2 to induce cardiomyocyte proliferation and compact zone expansion, and also acts on endothelial cells to<br />
support formation <strong>of</strong> the coronary vasculature.<br />
5.9 The role <strong>of</strong> Pod1/Tcf21 in epicardium-derived cells during cardiac development and fibrosis<br />
Caitlin M. Braitsch 1 , Michelle D. Combs 1 , Susan E. Quaggin 2 , and Katherine E. Yutzey 1<br />
1 Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA; 2 <strong>University</strong> <strong>of</strong> Toronto, Toronto, Ontario,<br />
Canada.<br />
During cardiac development, epicardium-derived cells (EPDCs) differentiate into fibroblasts and vascular<br />
smooth muscle (SM) cells. In the adult heart, EPDCs are reactivated upon cardiac injury. Immun<strong>of</strong>luorescence<br />
studies demonstrate that the transcription factors (TF) Pod1/Tcf21, WT1, Tbx18, and NFATC1 are expressed<br />
heterogeneously in EPDCs in chicken and mouse embryonic hearts. Expression <strong>of</strong> Pod1 and WT1, but not<br />
Tbx18 or NFATC1, is activated with all-trans-retinoic acid (RA) treatment <strong>of</strong> isolated avian EPDCs. In intact E7<br />
chicken hearts, RA signaling is required for full Pod1 expression and RA treatment inhibits SM differentiation.<br />
The requirements for Pod1 in differentiation <strong>of</strong> EPDCs in the developing heart were examined in mice lacking<br />
Pod1. Loss <strong>of</strong> Pod1 in mice leads to epicardial blistering, increased SM differentiation on the surface <strong>of</strong> the<br />
heart, and a paucity <strong>of</strong> interstitial fibroblasts, with neonatal lethality. On the surface <strong>of</strong> the myocardium,<br />
expression <strong>of</strong> multiple SM markers is increased in Pod1-deficient EPDCs, demonstrating premature SM<br />
differentiation. Together, these data demonstrate a critical role for Pod1 in controlling EPDC differentiation into<br />
SM during cardiac development. In adult hearts, cardiac injury leads to reactivation <strong>of</strong> fetal genes including<br />
WT1, Tbx18, and RALDH2 in EPDCs. Initial studies demonstrate that Pod1/Tcf21 expression is increased in<br />
mouse hearts subjected to myocardial infarction, transverse aortic constriction, or chronic isoproterenol infusion.<br />
Additional data will be presented describing Pod1/Tcf21 expression in mouse models <strong>of</strong> cardiac fibrosis.<br />
Together this work provides evidence for conserved Pod1/Tcf21-related regulatory mechanisms in EPDCs<br />
during cardiac development and fibrotic disease.<br />
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5.10 Epicardial GATA factors regulate coronary endothelial migration via Sonic hedgehog signaling.<br />
Kurt D. Kolander, Mary L. Holtz, and Ravi P. Misra<br />
Department <strong>of</strong> Biochemistry, Medical College <strong>of</strong> Wisconsin, Milwaukee, WI<br />
Early coronary vasculogenesis is dependent on the epicardium as a source <strong>of</strong> progenitor cells and regulatory<br />
signals. GATA transcription factors have been shown to be required for the formation <strong>of</strong> the proepicardium, a<br />
group <strong>of</strong> cells that give rise to the epicardium This suggests GATA factors may play a role in epicardial function.<br />
To address whether epicardial GATA-4 and GATA-6 may have a role in early coronary vascular development,<br />
we utilized a Wilm’s Tumor-1 promoter-driven Cre recombinase construct to conditionally knockout GATA-4 and<br />
GATA-6 in the epicardium. We found the combined loss <strong>of</strong> GATA-4 and GATA-6 resulted in embryonic lethality<br />
at E15.5, an age at which various coronary vascular defects commonly lead to lethality. Immun<strong>of</strong>luorescent<br />
imaging at E13.5 and E14.5 indicated an 80-90% decrease in sub-epicardial endothelial cells, which are<br />
required for coronary plexus formation. This led us to hypothesize that the decreased number <strong>of</strong> sub-epicardial<br />
endothelial cells is due to a loss <strong>of</strong> epicardial signaling to early endothelial or progenitor cells. Sonic hedgehog is<br />
an epicardial signaling factor required for the migration <strong>of</strong> sub-epicardial endothelial cells. Therefore, we<br />
immun<strong>of</strong>luorescently stained for Sonic hedgehog and found its expression was lost in the sub-epicardial space<br />
<strong>of</strong> the conditional knockout. These results are consistent with a novel role for epicardial GATA factors in<br />
coronary vascular development through regulation <strong>of</strong> a Sonic hedgehog signaling pathway<br />
Session 5: Valve Calcification & Development<br />
8.7 Analysis <strong>of</strong> TGFβ3 function in valve remodeling and aortic valve calcification<br />
Alejandra C Encinas, Robert Hinton # , and Mohamad Azhar<br />
Program in Developmental Biology and Neonatal Medicine, Herman B Wells Center for Pediatric Research,<br />
Indiana <strong>University</strong> School <strong>of</strong> Medicine, Indianapolis, IN; # Cincinnati Children's Hospital Medical Center,<br />
Cincinnati, OH<br />
Transforming Growth Factor beta (TGFβ) ligands function during the onset and progression <strong>of</strong> aortic valve<br />
calcification remains obscure as both contradictory elevated and suppressed TGFβ signaling has been found in<br />
patients <strong>of</strong> calcific aortic valve disease (CAVD). Here, using in vitro micromass cultures <strong>of</strong> mouse valve<br />
precursor cells (tsA58-AVM) we showed by complimentary realtime QPCR and synthetic luciferase reporter<br />
assays that TGFβ3 can upregulate Sry-related high-mobility-group box 4 (SOX4) transcription. Sox4 is initially<br />
expressed within the endocardially-derived tissue <strong>of</strong> both the outflow tract and atrioventricular canal during<br />
development, but persists within adult valve mesenchyme. Moreover, TGFβ3-dependent regulation <strong>of</strong> Sox4 can<br />
be blocked by a small molecule inhibitor <strong>of</strong> SMAD3 (SIS3), indicating that SMAD3 acts as a downstream<br />
mediator <strong>of</strong> TGFβ3-dependent Sox4 induction. Significantly, in situ hybridization revealed that TGFβ3 is the<br />
most prevalent TGFβ is<strong>of</strong>orm in postnatal heart valves. Histology demonstrated that bothTgfb3 -/- fetal (E14.5)<br />
and Smad3 -/- adult (6 month old) valves were abnormally thickened. There was no calcification in the aortic<br />
valves <strong>of</strong> Smad3 -/- mice. Correlative analysis <strong>of</strong> human valve tissues via immunohistochemistry (5 controls, 8<br />
CAVD, 4 non-calcific aortic valve disease) demonstrated diffused expression <strong>of</strong> SOX4 in the control tissues,<br />
which was extinguished in the diseased tissue specimens. Interestingly, the expression <strong>of</strong> SOX4 around the<br />
calcific nodules remains upregulated within the diseased aortic valves from CAVD patients. Consistently, Sox4<br />
siRNA knockdown in tsA58-AVM cells significantly decreased calcification. Overall, our results indicate that<br />
TGFβ3-dependent and SMAD3-mediated regulation <strong>of</strong> SOX4 may play an important role in valve remodeling<br />
and aortic valve calcification. Since small molecule inhibitor/s <strong>of</strong> SMAD3 could be used as a therapeutic<br />
approach, these data provide novel basic science rationale with potential to translate into different therapies for<br />
patients <strong>of</strong> CAVD.<br />
8.8 Nuclear exclusion <strong>of</strong> Sox9 in valve interstitial cells is associated with calcific phenotypes in heart<br />
valves<br />
Danielle J. Huk 1,2 , Harriet Hammond 2 , Robert. B. Hinton 3 , and Joy Lincoln 2<br />
1 Molecular and Cellular Pharmacology Graduate Program, <strong>University</strong> <strong>of</strong> Miami Miller School <strong>of</strong> Medicine, Miami,<br />
FL, 2 Center <strong>of</strong> Cardiovascular and Pulmonary Research, The Research Institute at Nationwide Children’s<br />
Hospital, Department <strong>of</strong> Pediatrics, The Ohio State <strong>University</strong>, Columbus, OH, 3 Division <strong>of</strong> Cardiology, The<br />
Heart Institute, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH,<br />
75
Calcific aortic valve disease (CAVD) is a major public health problem with no effective treatment available other<br />
than valve replacement surgery. Despite the clinical significance, the mechanisms <strong>of</strong> pathogenesis are poorly<br />
understood. Normal valve structures are composed <strong>of</strong> organized layers <strong>of</strong> extracellular matrix interspersed with<br />
valve interstitial cells (VICs) that share molecular phenotypes with cartilage, including Col2a1. In contrast,<br />
calcified valves are characterized by mineralized matrix and increased expression <strong>of</strong> osteogenic genes including<br />
Spp1. We have shown that Sox9 is required in vivo to promote the cartilaginous matrix and prevent calcification<br />
<strong>of</strong> healthy valves by directly activating Col2a1 and suppressing Spp1 in the nucleus. In this study, we show that<br />
in aortic valves from non-diseased subjects, Sox9 expression is restricted to the nuclei <strong>of</strong> VICs, while nuclear<br />
expression is downregulated and notably cytoplasmic in tissue collected from CAVD patients. Usin g a<br />
pharmacological approach, we show that Sox9-mediated calcification as a result <strong>of</strong> nuclear exclusion can be<br />
induced by retinoic acid (RA) treatment in vivo and in vitro. This decrease in nuclear Sox9 localization is<br />
associated with decreased Col2a1 expression and transactivation, activation <strong>of</strong> the Gene Ontology ‘bone<br />
development’ program and matrix mineralization. Ongoing studies include defining the signaling pathways that<br />
maintain Sox9 nuclear localization to prevent CAVD in healthy valves. Together, these data suggest that Sox9<br />
nucleocytoplasmic shuttling <strong>of</strong> Sox9 plays a role in valve pathogenesis and provides insights to aid in the<br />
development <strong>of</strong> new therapeutic strategies for CAVD.<br />
8.9 Runx2 is<strong>of</strong>orms have divergent roles in development and pathology in the heart<br />
Andre L.P. Tavares, Jesse A. Brown, Katarina Dvorak and Raymond B. Runyan<br />
Department <strong>of</strong> Cellular and Molecular Medicine, <strong>University</strong> <strong>of</strong> Arizona, Tucson, Arizona, U.S.A. 85724<br />
Formation <strong>of</strong> atrioventricular (AV) valves in the heart begins with an epithelial-mesenchymal cell transition<br />
(EMT). In the exploration <strong>of</strong> EMT, we investigated the presence <strong>of</strong> the transcription factor, Runx2, in the AV<br />
canal. Immunostaining and PCR showed that expression coincided with the onset <strong>of</strong> EMT. siRNA treatment <strong>of</strong><br />
AV canal cultures established that it was required for EMT and that loss specifically prevented the early cell<br />
separation step <strong>of</strong> EMT. Sequence analysis showed that the Runx2 is<strong>of</strong>orm present was Runx2-I and that this<br />
is<strong>of</strong>orm was regulated buy TGFβ2 but not BMP. Inhibition <strong>of</strong> Runx2-I with siRNA produced a loss <strong>of</strong><br />
mesenchymal cell markers. Comparison with siRNA towards Snail2, suggests that the two EMT transcription<br />
factors diverge in their regulation <strong>of</strong> downstream components. To determine whether the activity <strong>of</strong> Runx2-I was<br />
conserved in other EMT events, we examined human tissue samples in the progression <strong>of</strong> esophageal<br />
adenocarcinoma. Runx2-I was e xpressed coincident with the transition from columnar epithelia (Barrett’s<br />
Esophagus) to dysplasia, suggesting a conserved role in early EMT. The Runx2-II is<strong>of</strong>orm is associated with<br />
bone and calcifying tissues. To compare is<strong>of</strong>orm activities, we examined Runx2 is<strong>of</strong>orm expression in whole<br />
valve cultures from chicks and in micromass cultures <strong>of</strong> mouse valvular mesenchymal cells. Runx2-II inceases<br />
in culture consistent with expression <strong>of</strong> calcification markers including osteopontin and matrix gla protein and<br />
with positive staining for calcium. Together, the data show a requirement for TGFβ-regulated Runx2-I in EMT<br />
and BMP-regulated Runx2-II with calcification and calcified tissues. Supported by NHLBI HL82851 and Edwards<br />
Life Sciences.<br />
8.10 3D cardiac valve tissue reconstruction and quantitative evaluations <strong>of</strong> tissue & cell phenotypes in<br />
the Pdlim7 knock-out mouse.<br />
Rudyard W. Sadleir 1 , Robert W. Dettman 1 , Jennifer Krcmery 1 , Brandon Holtrup 1 , and Hans-Georg Simon 1<br />
1<br />
Department <strong>of</strong> Pediatrics, Northwestern <strong>University</strong> Feinberg School <strong>of</strong> Medicine, and Children’s Memorial<br />
Research Center, <strong>Chicago</strong>, IL, USA<br />
The actin binding protein Pdlim7 is part <strong>of</strong> a family <strong>of</strong> proteins involved in regulating actin dynamics. Pdlim7 is<br />
expressed throughout cardiac development and into adulthood, and inactivation in zebrafish and mouse models<br />
results in cardiac valve malformations. A recent model for atrio-ventricular (AV) valve formation and maturation<br />
describes the differential contribution <strong>of</strong> epicardially- and endocardially-derived cells to specific leaflets <strong>of</strong> the<br />
mouse valvuloseptal complex. Using the Pdlim7 knock-out mouse, we investigated whether the aberrant<br />
morphology <strong>of</strong> the adult mitral valve is caused by the distinct lineages that shape the lateral and septal leaflets.<br />
We integrate 3D heart valve reconstructions with novel methods to quantitatively test morphological differences<br />
between wildtypes and mutants. The quantitative analysis <strong>of</strong> valve shape demonstrates that our method can<br />
identify subtle, statistically significant morphological changes following loss <strong>of</strong> Pdlim7. From Pdlim7’s role in<br />
actin dynamics, it is possible that the valve tissue difference is the result <strong>of</strong> aberrant cellular migration and/or<br />
cellular shape. To investigate this, we present a 3D method developed to measure cell migration behavior, and<br />
introduce the field <strong>of</strong> 3D geometric morphometrics to studies <strong>of</strong> morphogenesis and statistically evaluate the<br />
76
patterns <strong>of</strong> cell shape diversity. This work in mice suggests Pdlim7 functions in AV valve remodeling and<br />
maturation at late embryonic and postnatal stages corresponding with the arrival <strong>of</strong> cells from the epicardial<br />
lineage. These findings are important because they describe a mouse model that links a novel gene (Pdlim7) to<br />
post-natal valve maturation and growth.<br />
Session 6: Stem Cells<br />
6.7 Dynamic Mesp1 regulation directs cardiac myocyte formation in embryonic stem cells<br />
Yu Liu 1 , Xueping Xu 2 , Austin Cooney 2 , Robert Schwartz 1<br />
Department <strong>of</strong> Biology and Biochemistry, <strong>University</strong> <strong>of</strong> Houston<br />
Department <strong>of</strong> Molecular and Cellular Biology, Baylor College <strong>of</strong> Medicine<br />
Mesp1 sits on the top <strong>of</strong> the hierarchy <strong>of</strong> cardiac gene regulation network. It emerges at the onset <strong>of</strong> mesoderm<br />
formation and disappears at the cardiac crescent. The extracellular cue that triggers Mesp1, and how the<br />
Mesp1-expressing cells differentiate into cardiovascular cell lineages are largely unknown. In this study, we<br />
have crossed the Mesp1 Cre/+ and Rosa EYFP/EYFP mouse strains, and established an ES cell line with the genotype<br />
Mesp1 Cre/+ /Rosa EYFP/+. By isolating EYFP (+) cells on day 4 <strong>of</strong> an ES differentiation protocol, we characterized<br />
the early cardiac progenitors. These cardiac progenitors enriched cardiac mesoderm markers (Flk1, PDGFRa,<br />
dHand), cardiac transcription factors (Nkx2-5, Tbx5, Mef2c), and excluded pluripotent markers (Oct4, Sox2,<br />
Nanog) and nascent mesoderm markers (T, Fgf8). Among a group <strong>of</strong> growth factors, BMP2/4 greatly induced<br />
the prevalence <strong>of</strong> EYFP (+) cells, while Wnt3a and Activin had only marginal effects. EYFP(+) cells represented<br />
a sub-population <strong>of</strong> the Flk1(+)/PDGFRa(+) cells, which were previously demonstrated to differentiate into<br />
cardiac lineages. Surprisingly, short period <strong>of</strong> BMP4 treatment (day 0-2) led to the induction <strong>of</strong> EYFP(+) cells<br />
and subsequent cardiac myocyte formation, whereas extended BMP4 (day 0-4) led to even more EYFP (+) cells<br />
but without subsequent cardiac myocyte formation. By introducing a pCAGG-loxP-bgeo-polyA-loxP-Mesp1<br />
cassette into this ES cell line, we achieved extended Mesp1 expression in EYFP(+) cells beyond the point that<br />
endogenous Mesp1 disappears, and proved that extended Mesp1 expression impaired cardiac myocyte<br />
formation. In summary, we have established an ES cell line that allows us to trace the fate <strong>of</strong> Mesp1-expressing<br />
cardiac progenitors. Using this line as the tool, we demonstrate that dynamic Mesp1 regulation directs cardiac<br />
myocyte formation in ES cells.<br />
6.8 Human amniocytes contain subpopulations <strong>of</strong> stem cells that have a repressed cardiogenic status.<br />
Colin T. Maguire, Bradley Demarest, Jennifer Akiona, Jonathon Hill, James Palmer, *Arthur R. Brothman, H.<br />
Joseph Yost, Maureen L. Condic<br />
Departments <strong>of</strong> Neurobiology and Anatomy and *Pediatrics and ARUP Laboratories, <strong>University</strong> <strong>of</strong> Utah<br />
Molecular Medicine Program, Eccles Institute <strong>of</strong> Human Genetics, Building 533, Room 3160, 15 North 2030<br />
East, Salt Lake City, Utah 84112-5330, USA<br />
Amniocytes are an intriguing candidate source <strong>of</strong> stem cells for autologous repair <strong>of</strong> congenital heart defects<br />
(CHD) in humans. Despite mesodermal fate poising, amniocytes are resistant to differentiating into fully<br />
functional cardiomyocytes. Here, we provide evidence that cultured amniocytes are a heterogeneous<br />
subpopulation <strong>of</strong> stem cells expressing key pluripotency and self-renewal markers. Interestingly, qPCR analysis<br />
<strong>of</strong> 17 independent amniocyte isolates also detected the expression <strong>of</strong> the core cardiogenic factors Mef2c, Tbx5,<br />
and Nkx2-5, as well as the cardiac chromatin regulator Baf60c (Smarcd3). Gata4 is not expressed, but some<br />
individual isolates show signs <strong>of</strong> a low level <strong>of</strong> gene leakiness. However, Tbx5 and the early-to-mid cardiac<br />
markers Mesp1, Isl1, and Gata6 were consistently detectable by both qPCR and immunostaining. Stimulating<br />
amniocytes with signals known to promote differentiation <strong>of</strong> cardiac lineages failed to upregulate mRNA<br />
transcript or protein levels <strong>of</strong> core cardiogenic factors or generate cells that stain positive for cardiac troponin T<br />
(CTNT) and MF20 markers. To more precisely define the cardiogenic status <strong>of</strong> amniocytes, we performed next<br />
generation RNA-seq analysis on amniocytes. Genome-wide expression pr<strong>of</strong>iling reveals a paradoxical<br />
molecular signature <strong>of</strong> cardiac markers, indicating that the developmental status <strong>of</strong> amniocytes as true cardiac<br />
progenitors is incomplete, yet uniquely poised. Amniocytes strongly express multiple ES cell and mesodermal<br />
repressors that may prevent cells from progressing further down a cardiogenic lineage. These results indicate<br />
that despite an incomplete cardiogenic phenotype, direct transdifferentiation and gene knockdown approaches<br />
may allow us to overcome these barriers and drive them to a mature cardiac cell fate.<br />
77
Supported in part by Primary Children’s Medical Foundation, T32HL007576 Cardiovascular Training Grant, and<br />
U01HL098179 (NHLBI Bench-to-Bassinet program)<br />
6.9 The Homeobox Transcription Factor, Irx4, Identifies a Mutipotent, Ventricular-specific Cardiac<br />
Progenitor<br />
Daryl Nelson 1 , Joshua Trzebiatowski 1 , Erin Willing 1 , Pearl Hsu 2 , Kevin Schoen 1 , Kevin Liberko 1 , Matthew<br />
Butzler 1 , Pat Powers 2 , Manorama John 2 , Timothy Kamp 1 , Gary Lyons 1<br />
1<br />
School <strong>of</strong> Medicine and Public Health, <strong>University</strong> <strong>of</strong> Wisconsin-Madison<br />
2<br />
<strong>University</strong> <strong>of</strong> Wisconsin Biotechnology Center<br />
Cardiac progenitors have been presented as potential cell therapeutics, due to their cardiogenic potency.<br />
Iroquois homeobox protein 4 (Irx4) is the earliest known marker <strong>of</strong> ventricular myocardium differentiation, and<br />
the transcription factor is restricted to the ventricles throughout embryogenesis and into adulthood. Our goal is<br />
to identify a multipotent, ventricular-specific cardiac progenitor. We have targeted the 3’ end <strong>of</strong> the Irx4 locus<br />
using a recombineering approach to insert fluorescence (mCherry), bioluminescence (luciferase), and antibiotic<br />
resistance (hph) cassettes. Six mESC clones were properly targeted Irx4 luc-mCh-hph/wt<br />
ES cells. RT-PCR, western<br />
blot analysis, and immun<strong>of</strong>luorescence assays demonstrated the functional integration <strong>of</strong> the reporters during<br />
embryoid body (EB) differentiation. Following 4 days <strong>of</strong> differentiation <strong>of</strong> the Irx4 luc-mCh-hph/wt cells in EBs,<br />
selection with hygromycin was carried out for 2 days, and day 6 cells were plated onto STO cell feeder layers for<br />
expansion. Selected Irx4 + cells are proliferative, expressing the cell cycle antigen, Ki67, and could be<br />
passaged more than 12 times. The Irx4 + cells express cKit, Flk1, and CXCR4 on the cell surface. Selected<br />
Irx4 + cells demonstrate cardiovascular potency when re-aggregated and differentiated in hanging drops resulted<br />
in ventricular myocyte-enriched cell preparations (65±3.7% cTnT + ; 67±2.6% Myl2 + , 22±3.1% SmMHC + , and<br />
6±2% CD31 + cells. The selected Irx4 + population represents the first ventricular-specific multipotent progenitor<br />
population identified and holds promise for generating all cardiovascular lineages necessary to reconstitute<br />
infarcted myocardium.<br />
6.10 Modeling the vascular phenotype <strong>of</strong> Williams-Beuren syndrome using induced pluripotent stem<br />
cells<br />
Caroline Kinnear, Wing Y. Chang, Shahryar Khattak, Karen Kennedy, Naila Mahmut, Tadeo Thompson,<br />
Aleksander Hinek, Gordon Keller, William L. Stanford, James Ellis, Seema Mital.<br />
Hosp for Sick Children, Toronto, ON, Canada<br />
Elastin is essential for arterial morphogenesis. Elastin haploinsufficiency in Williams-Beuren syndrome (WBS)<br />
leads to increased vascular smooth muscle cell (SMC) proliferation and vascular stenoses. We investigated the<br />
role <strong>of</strong> elastin in proliferation and differentiation <strong>of</strong> SMCs derived from human embryonic stem cells (hESCs),<br />
and WBS patient-derived induced pluripotent stem cells (hiPSCs). Human iPSCs were reprogrammed from skin<br />
fibroblasts from a WBS patient and BJ healthy control using 4-factor retrovirus reprogramming. hESCs and<br />
hiPSCs underwent directed differentiation to generate smooth muscle cells (SMCs). Differentiated cells were<br />
treated with a peptide or an anti-proliferative drug for 6 days. mRNA and protein expression <strong>of</strong> elastin, smooth<br />
muscle α-actin (SMA), Ki67 (proliferation marker) was assessed by high-content imaging, qRT-PCR, and flow<br />
cytometry. Vascular tube formation was assessed by 3D matrigel assays. Treatment <strong>of</strong> hESC-derived SMCs<br />
increased elastin expression, increased SMA+ cells, and reduced SMC proliferation. The reprogrammed<br />
fibroblasts expressed pluripotency genes and differentiated into all germ layers. SMC differentiation was<br />
confirmed by SMA, SM22a, myocardin, smoothelin expression and by contractile response to carbachol. When<br />
compared to BJ SMCs, WBS SMCs showed lower expression <strong>of</strong> elastin protein and mRNA which increased with<br />
treatment. WBS cells showed impaired SMC differentiation, increased SMC proliferation, and impaired vascular<br />
tube formation compared to BJ cells. Treatment with an elastin ligand and a candidate drug reduced SMC<br />
proliferation, enhanced SMC differentiation, and enhanced tube formation. In conclusion, WBS iPSCs<br />
demonstrated increased SMC proliferation and impaired SMC differentiation which was partially ameliorated<br />
with treatment. Our study provides an in vitro platform for modeling vascular disorders and future drug screens.<br />
78
General Poster Sessions<br />
Section 1: Human Genetics & CHD Models<br />
S1.1 Deletion <strong>of</strong> the extracellular matrix protein Adamtsl2 results in a inter-ventricular septal defect in<br />
mice<br />
Dirk Hubmacher, Lauren W.Wang, Suneel S. Apte<br />
Department <strong>of</strong> Biomedical Engineering, Lerner Research Institute, Cleveland Clinic<br />
Geleophysic dysplasia (GD) is a rare human genetic disorder presenting with short stature, joint contractures<br />
and thick skin. High morbidity, and frequently, juvenile mortality results from cardiac valvular and tracheopulmonary<br />
anomalies. Mutations in the extracellular proteins ADAMTSL2 or fibrillin-1 lead to recessive or<br />
dominant GD, respectively. Excess TGFβ signaling was described in cells derived from GD patients and may<br />
constitute a major pathogenetic mechanism. ADAMTSL2 directly interacts with latent transforming growth factorβ<br />
binding protein (LTBP)-1 and fibrillin-1. Fibrillin-1 and -2 assemble into micr<strong>of</strong>ibrils and modulate extracellular<br />
TGFβ and BMP signaling. The Adamtsl2 gene was deleted in mice by an intragenic IRES-lacZ-neomycin<br />
cassette, which also provided an expression reporter. Adamtsl2 deletion resulted in neonatal lethality due to an<br />
inter-ventricular septal defect (VSD) and lung abnormalities. β -gal reporter staining showed Adamtsl2<br />
expression within the embryonic heart and lung, consistent with the observed anomalies. Cardiac expression<br />
was primarily localized to the crest <strong>of</strong> the inter-ventricular septum, the atrio-ventricular valve annulus and<br />
coronary vessels. Histology revealed a VSD in 80% <strong>of</strong> the mice with enhanced, localized fibrillin-1 and -2<br />
staining at the crests <strong>of</strong> the inter-ventricular septum. In vitro, recombinant ADAMTSL2 bound to both fibrillin-1<br />
and -2, and accelerated the biogenesis <strong>of</strong> fibrillin-1 micr<strong>of</strong>ibril formation in fetal bovine nuchal ligament cells,<br />
while blocking fibrillin-2 assembly. This suggests that ADAMTSL2 may provide a mechanism to actively regulate<br />
the ratio <strong>of</strong> fibrillin-1 to fibrillin-2 in micr<strong>of</strong>ibrils, with potential consequences for TGFβ/BMP signaling. These<br />
studies suggest a new role for extracellular matrix in regulation <strong>of</strong> cardiac development.<br />
S1.2 Complex trait analysis <strong>of</strong> ventricular septal defects caused by Nkx2-5 mutation<br />
Claire E. Schulkey, Suk D. Regmi, Patrick Y. Jay<br />
Departments <strong>of</strong> Pediatrics and Genetics, Washington <strong>University</strong> School <strong>of</strong> Medicine, St. Louis, MO.<br />
Congenital heart disease is a complex trait. The same deleterious mutation typically causes widely varying<br />
presentations because additional, poorly defined factors modify phenotype. Historically, the modifiers have<br />
received less scrutiny than the causes. Nevertheless, the factors, especially ones that reduce risk, may suggest<br />
preventive strategies that a focus on monogenic causes has not. Hence, we assessed the role <strong>of</strong> genetic and<br />
environmental factors on the incidence <strong>of</strong> ventricular septal defects (VSD) caused by an Nkx2-5 heterozygous<br />
knockout mutation. We phenotyped >3100 hearts from a second generation intercross <strong>of</strong> the inbred mouse<br />
strains C57BL/6 and FVB/N. Genetic linkage analysis mapped loci with LOD scores <strong>of</strong> 5-7 on chromosomes 6, 8<br />
and 10 that influence the susceptibility to membranous VSD in Nkx2-5 +/- animals. The chromosome 6 locus<br />
overlaps one for muscular VSD susceptibility. Multiple logistic regression analysis for environmental variables<br />
revealed that maternal age is correlated with the risk <strong>of</strong> membranous and muscular VSD in Nkx2-5 +/- but not wildtype<br />
animals. The maternal age effect is unrelated to aneuploidy or a genetic polymorphism in the progeny.<br />
Ovarian transplant experiments between young and old females indicate that the basis <strong>of</strong> the maternal age effect<br />
resides in the mother and not the oocyte. Experiments to characterize a potential metabolic basis <strong>of</strong> the effect<br />
strongly suggest that the VSDs caused by Nkx2-5 mutation can be prevented. Enumerable factors contribute to<br />
the presentation <strong>of</strong> a congenital heart defect. Their characterization in a mouse model <strong>of</strong>fers the opportunity to<br />
define unanticipated pathways and to develop strategies for prevention.<br />
S1.3 Functional analysis <strong>of</strong> novel ZIC3 mutations identified in patients with heterotaxy<br />
Jason Cowan, Muhammad Tariq, Stephanie M. Ware<br />
<strong>University</strong> <strong>of</strong> Cincinnati<br />
Loss <strong>of</strong> function mutations in the zinc finger in cerebellum 3 (ZIC3) transcription factor result in heterotaxy, a<br />
condition characterized by abnormal left-right positioning <strong>of</strong> thoraco-abdominal organs and a wide variety <strong>of</strong><br />
congenital anomalies, particularly <strong>of</strong> the cardiovascular system. Mutations in ZIC3 have been reported in<br />
approximately 75% <strong>of</strong> all familial and 1% <strong>of</strong> all sporadic heterotaxy cases and have been associated with<br />
79
VACTERL, a constellation <strong>of</strong> malformations phenotypically overlapping heterotaxy. The presence <strong>of</strong> a<br />
polyalanine (polyA) expansion in one patient with VACTERL is <strong>of</strong> particular interest as pathogenic expansions<br />
have been identified in several other developmentally critical transcription factors, including ZIC2. To further<br />
define the incidence and functional significance <strong>of</strong> ZIC3 mutations in heterotaxy, coding regions and splice<br />
junctions were screened in 200 unrelated heterotaxy patients. Nine mutations (8 novel) were identified,<br />
including a single alanine expansion (c.insGCC159-160). Functional analyses were supplemented by 4<br />
additional recently reported ZIC3 mutations. Aberrant ZIC3 cytoplasmic localization was observed for mutations<br />
spanning multiple nuclear localization domains between amino acids 155 and 318 and correlated with<br />
decreased transactivation <strong>of</strong> a luciferase reporter. A missense mutation within zinc finger 5 (p.A447G)<br />
surprisingly increased luciferase transactivation, despite elevated levels <strong>of</strong> cytoplasmic ZIC3. Neither polyA tract<br />
expansion differed significantly from wildtype with respect to either luciferase transactivation or ZIC3 subcellular<br />
localization. These analyses collectively indicate a higher than expected percentage <strong>of</strong> ZIC3 mutations in<br />
patients with sporadic heterotaxy and suggest alternative pathogenesis <strong>of</strong> some ZIC3 mutations, notably those<br />
within the polyA tract.<br />
S1.4 The Genetic Basis <strong>of</strong> Isolated Tetralogy <strong>of</strong> Fallot<br />
Marcel Grunert 1,2,* , Cornelia Dorn 1,2,3,* , Markus Schueler 1,2 , Ilona Dunkel 1 , Jenny Schlesinger 2 , Siegrun Mebus 4 ,<br />
Katherina Bellmann 2,3 , Vladimir Alexi-Meskishvili 5 , Sabine Klaassen 6 , Katharina Wassilew 7 , Bernd<br />
Timmermann 8 , Roland Hetzer 5 , Felix Berger 4 , Silke R. Sperling 1,2,3<br />
1 Group <strong>of</strong> Cardiovascular Genetics, Max Planck Institute for Molecular Genetics, 2 Department <strong>of</strong> Cardiovascular Genetics,<br />
Experimental and Clinical Research Center, Charité – <strong>University</strong> Medicine Berlin and Max Delbrück Center for Molecular<br />
Medicine, 3 Department <strong>of</strong> Biology, Chemistry and Pharmacy at Freie Universität <strong>of</strong> Berlin, 4 Department <strong>of</strong> Pediatric<br />
Cardiology, 5 Department <strong>of</strong> Cardiac Surgery and 7 Department <strong>of</strong> Pathology at German Heart Center Berlin, 6 National<br />
Registry <strong>of</strong> Congenital Heart Disease, 8 Next Generation Service Group at Max Planck Institute for Molecular Genetics,<br />
* shared authorship<br />
Background: Tetralogy <strong>of</strong> Fallot (TOF) represents 10% <strong>of</strong> congenital heart disease, which are the most<br />
common birth defect in human. The majority <strong>of</strong> TOF are isolated non-syndromic cases <strong>of</strong> unknown precise<br />
cause, which applies for the majority <strong>of</strong> congenital heart disease. Methods: We performed targeted<br />
resequencing <strong>of</strong> over 1,000 genes and microRNAs as well as whole transcriptome and miRNome analysis in<br />
TOF cases, parents and controls using next-generation sequencing techniques. We defined a set <strong>of</strong> TOF genes<br />
with deleterious variations and which are mutated at a higher rate in TOF subjects compared to healthy controls.<br />
Results: We identified an oligogenic architecture underlying TOF, which discriminate TOF cases from controls.<br />
On average, TOF subjects show a combination <strong>of</strong> novel and inherited variations in five genes. The majority <strong>of</strong><br />
genes has known association with human cardiac disease and/or shows a cardiac phenotype in mouse mutants.<br />
Seven genes are novel and have not yet been linked to a cardiac phenotype. Functionally interacting yet<br />
individual mutations lead to the same phenotypic outcome during development. The majority <strong>of</strong> TOF genes<br />
shows continuous relevance during adulthood. Conclusions: Isolated TOF is a genetic disorder involving<br />
multiple genes. Although subjects show a range <strong>of</strong> individual mutations, the phenotypic outcome is similar<br />
because TOF genes show common patterns <strong>of</strong> functional interactions. Sequencing approaches can help to<br />
define a genetic risk pr<strong>of</strong>ile for families that can also be used to define differences in the long-term clinical<br />
outcome, which should permit a personalization <strong>of</strong> diagnostic and therapeutic strategies.<br />
S1.5 An Excess <strong>of</strong> Deleterious Variants in VEGF-A Pathway Genes in Down Syndrome-Associated<br />
Atrioventricular Septal Defects<br />
Christine Ackerman 1 , Adam E. Locke 2,8 , Eleanor Feingold 3 , Benjamin Reshey 1 , Karina Espana 1 , Janita<br />
Thusberg 4 , Sean Mooney 4 , Lora J. H. Bean 2 , Kenneth J. Dooley 5 , Clifford Cua 6 , Roger H. Reeves 7 , Stephanie L.<br />
Sherman 2 , Cheryl L. Maslen 1,*<br />
1 Division <strong>of</strong> Cardiovascular Medicine and the Heart Research Center, Oregon Health & Science <strong>University</strong>, Portland, OR<br />
97239<br />
2 Department <strong>of</strong> Human Genetics, Emory <strong>University</strong>, Atlanta, GA 30033<br />
3 Department <strong>of</strong> Human Genetics, Graduate School <strong>of</strong> Public Health, <strong>University</strong> <strong>of</strong> Pittsburgh, Pittsburgh PA, 15261<br />
4 Buck Institute for Research on Aging, Novato, CA 94945<br />
5 Sibley Heart Center Cardiology, Children’s Hospital <strong>of</strong> Atlanta, Atlanta, GA 30033<br />
6 Heart Center, Nationwide Children’s Hospital, Columbus, OH 43205<br />
7 Department <strong>of</strong> Physiology and the Institute for Genetic Medicine, Johns Hopkins <strong>University</strong> School <strong>of</strong> Medicine, Baltimore,<br />
MD 21205<br />
8 Current address: Center for Statistical Genetics, Department <strong>of</strong> Biostatistics, <strong>University</strong> <strong>of</strong> Michigan School <strong>of</strong> Public Health,<br />
Ann Arbor, MI 48109<br />
80
About half <strong>of</strong> people with trisomy 21 have a congenital heart defect (CHD) while the remainder have a<br />
structurally normal heart, demonstrating that trisomy 21 is a significant risk factor but is not causal for abnormal<br />
heart development. We used a candidate gene approach in a study cohort <strong>of</strong> individuals with Down syndrome<br />
(DS) to determine if rare genetic variants in genes involved in atrioventricular valvuloseptal morphogenesis<br />
contribute to atrioventricular septal defects (AVSD) in this sensitized population. We found a significant excess<br />
(p
future studies, we will test whether FA/MI supplementation rescues these defects through normalization <strong>of</strong><br />
cardiac function. Our contributions could be a first step in implementing new therapeutic strategies based on<br />
FA/MI prevention <strong>of</strong> birth defects. Research Support: HL083048, HL095717.<br />
1.8 Noonan syndrome associated RAF1 mutant evokes hypertrophic cardiomyopathy features in human<br />
cardiomyocytes in vitro.<br />
Malte Tiburcy 1 , Nicole Dubois 2 , Aleksander Hinek 3 , Seema Mital 3 , Darrell Kotton 4 , Wolfram Zimmermann 1 ,<br />
Gordon Keller 2 , Benjamin Neel 2 , Toshiyuki Araki 2<br />
1 <strong>University</strong> <strong>of</strong> Gottingen, 2 <strong>University</strong> Health Network, 3 Hospital for Sick Children, Boston <strong>University</strong> 4<br />
Noonan syndrome (NS) and its related disorders, which are now called “RASopathies”, are caused by aberrant<br />
activation <strong>of</strong> RAS/ERK pathway. Most RASopathies feature proportional short stature, facial dysmorphia,<br />
cognitive impairment and cardiac defects. The cardiac manifestations in RASopathies vary widely, but<br />
hypertrophic cardiomyopathy (HCM) is found in virtually all NS cases caused by RAF1 allele that encode a<br />
hyperactive kinase mutant. HCM also is common in LEOPARD syndrome and Costello syndrome. We reported<br />
previously that a mouse model <strong>of</strong> NS caused by a kinase-activated Raf1 mutant recapitulates major features <strong>of</strong><br />
NS, including HCM. Importantly, these features were normalized by post-natal treatment <strong>of</strong> MEK inhibitor. To<br />
extend these mouse studies to pre-clinical human models to better identify detail molecular basis and signaling<br />
as well as to aid in the development <strong>of</strong> new therapies for RASopathies, we have generated human induced<br />
pluripotent stem cells (hiPSCs) from fibroblasts <strong>of</strong> multiple RASopathy patients. We found that a kinaseactivating<br />
RAF1 mutant causes increased cell size <strong>of</strong> cardiomyocytes differentiated from hiPSCs compared with<br />
normal hiPSCs. We also found increased calcium sensitivity and lower response to b adrenergic stimulation<br />
when we cultured differentiated cardiomyocytes as engineered heart tissue, which allows us to apply<br />
mechanical forces on these cardiomyocytes. Our data show that NS associated RAF1 mutant can cause HCM<br />
phenotypes in vitro and provide a potential pre-clinical system for testing new therapies.<br />
1.9 Engineering new mouse models to map dosage-sensitive genes in Down syndrome congenital heart<br />
defects<br />
Eva Lana-Elola 1 , Sheona Watson-Scales 1 , Amy Slender 1 , Louisa Dunlevy 1 , Mike Bennett 1 , Timothy Mohun 1 ,<br />
Elizabeth M. C. Fisher 2 , Victor L. J. Tybulewicz 1<br />
1<br />
MRC, National Institute for Medical Research, London, UK<br />
2<br />
UCL Institute <strong>of</strong> Neurology, London, UK<br />
Trisomy <strong>of</strong> human chromosome 21 (Hsa21) occurs in ~1 in 750 live births and the resulting gene dosage<br />
imbalance causes Down syndrome (DS). One <strong>of</strong> the most important medical aspects are the congenital heart<br />
defects (CHD), which affect around half <strong>of</strong> all individuals with DS. The defects observed in DS range from<br />
simple chamber septation to complex CHD involving multiple aspects <strong>of</strong> the heart anatomy. The most common<br />
CHD in DS affect the atrio-ventricular (AV) junction. Using high-resolution episcopic microscopy (HREM) in<br />
mouse embryonic hearts, we have studied a ‘transchromosomic’ (Tc1) mouse model <strong>of</strong> DS, which carries<br />
Hsa21 as a freely segregating chromosome. The Tc1 mouse closely resembles human DS CHD, replicating<br />
many <strong>of</strong> the features <strong>of</strong> the AV septal defects found in DS (Dunlevy, 2010). To unravel the mechanisms <strong>of</strong><br />
defective heart formation in the Tc1 mouse embryo, we are using lineage markers to study the tissues involved<br />
in development <strong>of</strong> the AV junction. In order to identify dosage-sensitive genes that may underlie DS CHD and<br />
other complex biological effects <strong>of</strong> trisomy, we are generating a high-resolution mapping panel <strong>of</strong> mouse strains<br />
with partial trisomies and monosomies for regions <strong>of</strong> mouse chromosomes orthologous to Hsa21. Using<br />
systematic HREM analysis, we are examining partial trisomic strains to identify regions that are sufficient to<br />
induce the CHD phenotype. Conversely, by crossing Tc1 mice to partial monosomic strains and assaying the<br />
effect <strong>of</strong> each interval on the cardiac phenotype, we are testing individual genes for their potential role in DS<br />
CHD.<br />
1.10 Recovery <strong>of</strong> ENU induced mutations causing congenital heart disease using next-gen sequencing<br />
You Li, Sarosh Fatakia, Ashok Srinivasan, Histao Yagi, Rama Damerla, Bishwanath Chatterjee, Cecilia W. Lo<br />
Dept <strong>of</strong> Developmental Biology, <strong>University</strong> <strong>of</strong> Pittsburgh School <strong>of</strong> Medicine, Pittsburgh, PA<br />
Forward genetic screens provide a powerful non-gene biased approach to elucidate the genetic basis for<br />
congenital heart disease (CHD). In the past this has been hampered by difficulties in mutation recovery. In our<br />
current large-scale recessive mouse ENU mutagenesis screen, we are pursuing the use <strong>of</strong> whole<br />
genome/whole exome sequencing for mutation recovery. To evaluate the efficacy <strong>of</strong> our mutation recovery<br />
82
pipeline, the mutant Destro exhibiting complex CHD with heterotaxy was analyzed by both whole exome and<br />
whole genome sequencing. A multi-step bioinformatics filtering strategy was developed for mutation recovery.<br />
First, we removed sequence variants present in dbSNP129 or our in-house mouse databases. Second, coding<br />
variants likely to be homozygous based on sequence coverage, unique reads, and strandedness were<br />
prioritized as potential candidates. Third, mutations are further rank ordered based on cross-species<br />
conservation. Finally, the causal mutation is identified by genotyping multiple mutants with the same phenotype.<br />
Whole-genome sequencing <strong>of</strong> Destro with 8x coverage together with our filtering strategy reduced 178,848<br />
variants to just 6, with genotyping analysis identifying Bicc1 c.606+2T>C as the disease-causing mutation. This<br />
same mutation was also recovered by whole-exome sequencing with ~50x target-coverage. In another mutant<br />
line 370 with coarctation and diaphragmatic hernia, exome sequencing analysis yielded a single mutation in<br />
Lox1, which was confirmed to be the disease causing mutation. Over 10 mutations have been recovered thus<br />
far. These results suggest our whole exome-sequencing pipeline will provide a cost-effective strategy for<br />
meeting our goal <strong>of</strong> pursuing a saturation level mutagenesis screen to recover mutations causing CHD.<br />
Supported by funding from U01-HL098180<br />
1.11 Resource Sharing by the Cardiovascular Development Consortium: A basic research component<br />
<strong>of</strong> the NHLBI Bench to Bassinet Program<br />
Charlene Schramm 1 , Benoit G. Bruneau 2 , Cecilia Lo 3 , Jonathan Seidman 4 , H. Joseph Yost 5 , Sharon Tennstedt 6 ;<br />
for the Cardiovascular Development Consortium Investigators<br />
1<br />
National Heart, Lung, and Blood Institute, National Institutes <strong>of</strong> Health, Bethesda, MD<br />
2<br />
Gladstone Institute <strong>of</strong> Cardiovascular Disease, San Francisco, CA<br />
3<br />
Dept. <strong>of</strong> Developmental Biology, <strong>University</strong> <strong>of</strong> Pittsburgh, Pittsburgh, PA<br />
4<br />
Dept. <strong>of</strong> Genetics, Harvard Medical School, Boston, MA<br />
5<br />
Dept. <strong>of</strong> Neurobiology and Anatomy, <strong>University</strong> <strong>of</strong> Utah, Salt Lake City, UT<br />
6<br />
New England Research Institutes, Watertown, MA<br />
The NHLBI Cardiovascular Development Consortium (CvDC) is a collaborative association <strong>of</strong> four Research<br />
Centers that comprise the basic science component <strong>of</strong> the NHLBI Bench-to-Bassinet (B2B) Program. The roles<br />
<strong>of</strong> the CvDC within the B2B Program are two-fold; 1) to expand knowledge <strong>of</strong> the complex regulatory networks<br />
that affect cardiac development, and 2) through interaction with a sister consortium within the B2B Program, the<br />
Pediatric Cardiac Genomics Consortium, apply CvDC discoveries to clinically-relevant genomics studies <strong>of</strong><br />
human congenital heart disease. The role <strong>of</strong> the CvDC within the cardiovascular development community is to<br />
generate and rapidly share novel resources, including published and unpublished datasets, novel model<br />
organisms, reagents, and tools. As CvDC resources become available, they are assembled into an online<br />
collection accessible to the scientific community through a portal on the B2B Program website<br />
(http://www.benchtobassinet.org/Investigators.asp). The collection includes resources directed to the study <strong>of</strong><br />
the epigenetics <strong>of</strong> cardiac development, gene discovery <strong>of</strong> genetic variants and networks causative <strong>of</strong> heart<br />
defects in animal models, and transcriptome changes in response to genetic variants or epigenetic regulation.<br />
The portal provides descriptions <strong>of</strong> available resources, and links to data repositories or vendors. Data and<br />
bioinformatics tools, such as mouse and zebrafish ChIP-seq and RNA-seq datasets or completeMOTIFs, are<br />
accessible through the CvDC data repository (https://b2b.hci.utah.edu/gnomex/gnomexGuestFlex.jsp), while<br />
mouse models are available through Jackson Laboratories or directly from CvDC Research Centers.<br />
Alternatively, CvDC datasets can be searched or browsed directly on the data repository, and mouse lines on the<br />
Jackson Laboratories Mouse Genome Informatics website.<br />
1.12 Large scale forward genetic screen in fetal mice reveals genetic etiology for hypoplastic left heart<br />
syndrome and wide spectrum <strong>of</strong> congenital heart defects<br />
Xiaoqin Liu, Andrew J. Kim, Guozhen Chen, Richard Francis, Deborah Farkas, You Li, Wendy Shung, Sarosh<br />
Fatakia, Ashok Srinivasan, Shane Anderton, Heather Lynn, Youngsil Kim, Liyin Wang, Chien-fu Chang,<br />
Kimimasa Tobita, Linda Leatherbury, Cecilia W. Lo<br />
Dept <strong>of</strong> Developmental Biology, <strong>University</strong> <strong>of</strong> Pittsburgh School <strong>of</strong> Medicine, Pittsburgh, PA.<br />
We are conducting a large-scale mouse ENU mutagenesis screen using noninvasive mouse fetal<br />
echocardiography to recover mutations causing congenital heart disease (CHD). Ultrasound screening <strong>of</strong><br />
42,628 mouse fetuses yielded 2,430 abnormal fetuses. Of these, 1,614 (66.4%) exhibited CHD, with 43.5%<br />
dying prenatally. Growth retardation was highly associated with cardiac defects (88.3%). Over 80 mutant lines<br />
with heritable CHD phenotypes have been recovered; the most common being ventricular septal defect (VSD),<br />
83
found in 59 mutant lines. Double outlet right ventricle and atrioventricular septal defect were the most common<br />
complex CHD phenotypes observed. Surprisingly, 27 <strong>of</strong> the mutant lines exhibited laterality defects. We also<br />
recovered three mutant lines with hypoplastic left heart syndrome (HLHS), a phenotype not previously observed<br />
in any mouse models. Ultrasound phenotyping has high detection sensitivity for hypoplastic left and right heart<br />
syndromes, VSD, and persistent tru ncus arteriosus/pulmonary atresia, but low detection rate for coronary fistula<br />
and aortic arch anomalies. However, mutants with such defects can be recovered using micro-computed<br />
tomography as a secondary screening tool. Exome sequencing is underway to recover the disease causing<br />
mutations, with mutations identified in 11 mutant lines thus far. Lines with heritable CHD phenotypes have been<br />
cryopreserved at the Jackson Laboratory for public access. These lines are annotated in the Mouse Genome<br />
Informatics (MGI) database with detailed phenotype information provided in multiple imaging modalities. Clinical<br />
CHD Fyler codes are also curated to show human CHD correlates. These novel mouse models will greatly aid in<br />
elucidating the developmental etiology <strong>of</strong> HLHS and other CHD.<br />
Supported by U01-HL098180.<br />
1.13 Molecular genetic investigation <strong>of</strong> Left Ventricular Outflow Tract Obstruction<br />
Dr Catherine L Mercer 1 , Dr Ita O’Kelly 1 , Dr David Fitzpatrick 2 , Pr<strong>of</strong>essor David I Wilson 1<br />
1<br />
Centre for Human Development, Stem Cells and Regeneration, Faculty <strong>of</strong> Medicine, <strong>University</strong> <strong>of</strong><br />
Southampton, UK<br />
2<br />
MRC Human Genetics Unit, Western General Hospital, Edinburgh, UK<br />
Introduction: Left ventricular outflow tract obstruction (LVOTO) is a significant cause <strong>of</strong> morbidity and mortality<br />
and a strong genetic component is established. A patient has been identified with LVOTO, and a balanced<br />
translocation with breakpoints on 14q and 15q. This is suggestive <strong>of</strong> a disease causing gene within either<br />
region. Hypothesis: A gene within 14q or 15q is critical to cardiogenesis and mutations within it are a cause <strong>of</strong><br />
LVOTO in humans. Methods: Using mouse RNA Whole Mount In Situ Hybridisation, the embryonic expression<br />
pattern <strong>of</strong> murine orthologous genes within the 1 megabase region <strong>of</strong> the breakpoints were studied in<br />
developing mouse embryos to determine expression during cardiogenesis. Since genes involved in mammalian<br />
cardiogenesis are conserved, murine orthologs <strong>of</strong> human genes can usefully be screened in this way. Images<br />
were recorded using optical projection tomography. The expression patterns allowed prioritisation <strong>of</strong> genes to be<br />
selected for immunohistochemistry and sequencing. Results: Literature review for cases <strong>of</strong> 14q and 15q<br />
deletions and the associated phenotypes identified a region on 15q as the most likely locus. WISH shows<br />
expression <strong>of</strong> MCTP2 (15q26.2) in mouse embryonic hearts and using immunohistochemistry, MCTP2 was also<br />
found to localise to the sarcomeres <strong>of</strong> cardiomyocytes in human fetal hearts. Sequencing <strong>of</strong> MCTP2 in 53 cases<br />
<strong>of</strong> LVOTO looking for mutations within the coding sequence/splice sites revealed a missense mutation,<br />
p.Arg340Cys not found in 310 ethnically matched control chromosomes. Conclusions: MCTP2 is a good<br />
candidate disease gene for LVOTO.<br />
1.14 Exome Sequencing Analysis <strong>of</strong> a Pleiotropic Congenital Heart Disease Family<br />
Ashok Kumar Manickaraj 1 , Orion Buske 2 , Michael Brudno 1,2,3 , Seema Mital 1<br />
1<br />
Hospital for Sick Children, Toronto, ON, Canada<br />
2<br />
Department <strong>of</strong> Computer Science, <strong>University</strong> <strong>of</strong> Toronto, Toronto, ON, Canada<br />
3<br />
Donnelly Centre and the Banting and Best Department <strong>of</strong> Medical Research, <strong>University</strong> <strong>of</strong> Toronto,Toronto,<br />
ON, Canada<br />
Background – Non-syndromic congenital heart defects (CHD) have been observed in families underscoring<br />
possible single and multiple gene defects that are yet to be completely identified. Candidate gene and other<br />
approaches have limited sensitivity in detecting rare causal variants. We used next generation sequencing to<br />
identify disease-associated variants in a family with highly penetrant pleiotropic CHD. Methods and Results –<br />
Whole exomic sequencing performed on 4 members <strong>of</strong> a family with different CHD lesions using Illumina Hi-Seq<br />
2000 platform, yielded more than 1700 variants. After excluding common variants (1000 genomes, dbSNP) and<br />
sequencing artifacts, variants were prioritized based on cardiac and vascular expression <strong>of</strong> genes. Of the 55<br />
novel and rare variants (including single nucleotide variations and indels) seen in the 4 members, 49 were nonsynonymous,<br />
3 were predicted to alter splicing, and 3 resulted in a frameshift. Further prioritization reduced the<br />
number <strong>of</strong> variants potentially involved in CHD to 5. Of those 5, a novel non-synonymous variant on<br />
chromosome 4 segregated in affected family members i.e. the proband with interupted aortic arch, proband’s<br />
brother with ventricular septal defect, father with patent ductus arteriosus, but not in mother with no heart<br />
disease. The mutation has a good SIFT prediction <strong>of</strong> 0.01. Mice knockouts have previously shown this gene is<br />
essential for embryonic hematopoiesis including heart development. Conclusions – Next-generation<br />
84
sequencing can help identify novel and rare variants associated with CHD. We will study the expression <strong>of</strong> this<br />
gene in cardiac tissue available in the proband and replicate in additional CHD families.<br />
1.15 High prevalence <strong>of</strong> left-right patterning mutants recovered in a large scale mouse mutagenesis<br />
screen for mutations causing congenital heart defects<br />
Deborah Farkas, Richard Francis, You Li, Shane Anderton, Heather Lynn, Youngsil Kim, Liyin Wang, Kimimasa<br />
Tobita, Linda Leatherbury, Cecilia W. Lo<br />
Dept <strong>of</strong> Developmental Biology, <strong>University</strong> <strong>of</strong> Pittsburgh School <strong>of</strong> Medicine, Pittsburgh, PA.<br />
Some <strong>of</strong> the most complex congenital heart disease (CHD) is found in conjunction with heterotaxy (HTX), the<br />
randomized left-right patterning <strong>of</strong> visceral organ situs. In our ongoing large scale mouse mutagenesis screen<br />
for mutations causing congenital heart disease, more than 1/3 <strong>of</strong> the mutant lines recovered from our screen<br />
exhibited left-right patterning defects. This would suggest an important role for pathways involved in left-right<br />
patterning in cardiovascular morphogenesis, perhaps not unexpected given the heart is the most left-right<br />
asymmetric organ in the body. From over 80 mutant lines recovered thus far in our screen, 27 display laterality<br />
defects (37%). Of these, 14 exhibit either complete mirror symmetric visceral organ situs (situs inversus totalis;<br />
SIT) or HTX, 10 lines displaying only HTX, and 3 lines with only SIT. Importantly, heterotaxy mutants almost<br />
always have complex CHD, but complex CHD is never observed in mutants with SIT. In mutants where the mut<br />
ations have been identified, genotyping have revealed some mutant animals have normal situs (situs solitus)<br />
and such animals never have complex CHD. Given the importance <strong>of</strong> motile cilia function in laterality<br />
specification, we also carried out videomicroscopy <strong>of</strong> tracheal airway cilia as a proxy for nodal cilia motility in<br />
mutant lines exhibiting laterality defects. We have found mutants exhibiting a wide range <strong>of</strong> ciliary phenotypes<br />
ranging from normal cilia motility, to those with slow and dyskinetic ciliary beat, hyperkinetic ciliary beat, or<br />
immotile cilia. Interestingly, as we have reported previously, mutants with immotile cilia can also generate<br />
mutants with normal situs solitus or SIT, suggesting motile cilia function may be dispensible for left-right<br />
patterning. By systematically categorizing the situs defect and CHD phenotypes observed in laterality mutants<br />
recovered in our mutagenesis screen, we hope to ascertain the discrete steps involved in visceral organ situs<br />
specification, and elucidate the role <strong>of</strong> cilia in left-right patterning and cardiac morphogenesis.<br />
Supported by U01-HL098180.<br />
1.16 The National Registry <strong>of</strong> Genetically Triggered Thoracic Aortic Aneurysms (GenTAC): Registry<br />
Progress and Research Successes<br />
Cheryl L. Maslen, PhD, on behalf <strong>of</strong> the GenTAC Consortium<br />
Funded by the National Institutes <strong>of</strong> Health, the GenTAC Registry is a multicenter, longitudinal, observational<br />
cohort study <strong>of</strong> patients with conditions involving genetically triggered thoracic aortic aneurysm and/or dissection<br />
(TAAD). GenTAC was established to provide a biospecimen inventory and bioinformatics infrastructure that will<br />
enable research to advance the clinical management <strong>of</strong> such patients. Primary diagnoses include Marfan<br />
syndrome, bicuspid valve with aneurysm and/or a family history <strong>of</strong> aneurysm, idiopathic TAAD in patients < 50<br />
years <strong>of</strong> age, Turner syndrome, familial TAAD, other congenital heart disease with TAAD, Ehlers-Danlos<br />
syndrome, and Loeys-Dietz syndrome. To date GenTAC has recruited 2826 subjects, with a final goal <strong>of</strong> 4000<br />
registrants. Initial analyses <strong>of</strong> GenTAC data/specimens have included studies <strong>of</strong> genetic causes for aortic<br />
syndromes via gene sequencing, SNP, CNV and genome-wide association studies, potential usefulness <strong>of</strong><br />
TGF-b blood levels as prognostic or therapeutic marker in Marfan subjects, surgical approaches/outcomes for<br />
ascending aortic conditions and gender differences in TAAD. Other studies in progress include cross-sectional<br />
and longitudinal data regarding phenotype-genotype correlations <strong>of</strong> disease risk factors, features, treatment,<br />
and outcomes, and analysis <strong>of</strong> imaging methods/integration <strong>of</strong> imaging findings with clinical and genetic data.<br />
GenTAC phenotyping and imaging cores have been established to facilitate these inquiries. The GenTAC<br />
Registry is a resource for anyone in the scientific community interested in advancing our understanding <strong>of</strong><br />
genetically mediated TAAs and their causes, diagnosis, and optimal treatment. Investigators interested in<br />
utilizing GenTAC for ancillary studies should contact the registry at gentac-registry@rti.org or apply at<br />
http://gentac.rti.org.<br />
1.17 High-throughput phenotyping for structural heart defects in fetal/neonatal mice using microcomputed<br />
tomography<br />
Andrew J. Kim 1 , Richard Francis 1 , Wendy Shung 1 , Xiaoqin Liu 1 , Guozhen Chen 1 , William A. Devine 1,2 , Deborah<br />
R. Farkas 1 , Linda Leatherbury 3 , Kimimasa Tobita 1,2 , Cecilia W. Lo 1<br />
1 Department <strong>of</strong> Developmental Biology, <strong>University</strong> <strong>of</strong> Pittsburgh School <strong>of</strong> Medicine, Pittsburgh, PA<br />
85
2 Children’s Hospital <strong>of</strong> Pittsburgh <strong>of</strong> the <strong>University</strong> <strong>of</strong> Pittsburgh Medical Center, Pittsburgh, PA<br />
3 Children’s National Medical Center, Washington, DC<br />
We are conducting a high-throughput mouse mutagenesis screen to recover mutations causing congenital heart<br />
defects (CHD) using fetal echocardiography. Mutant fetuses identified to have cardiac defects by ultrasound<br />
must be further phenotyped by necropsy and histological analysis for CHD diagnosis. In this study, we<br />
investigated the utility <strong>of</strong> micro-computed tomography (micro-CT) as a high-throughput screening tool for<br />
identifying structural heart defects in fetal/newborn mice. Micro-CT scanning <strong>of</strong> 2105 fetal/newborn mice carried<br />
out with iodine contrast agent revealed a spectrum <strong>of</strong> CHD, the most common being ventricular septal defects<br />
(VSDs; 14.58%), followed by outflow tract anomalies (2.6%), including transposition <strong>of</strong> the great arteries (TGA;<br />
0.67%), double outlet right ventricle (DORV; 1.71%), pulmonary atresia (PA; 0.10%), and persistent truncus<br />
arteriosus (0.14%). We also observed right aortic arch (RAA; 1.33%), coarctation/interrupted aortic arch<br />
(0.57%), endocardial cushion defects (1.05%) and tricuspid atresia/stenosis (0.62%). These same specimens<br />
were evaluated by necropsy, with some further analyzed by episcopic fluorescence image capture (EFIC). EFIC<br />
generates serial 2D histological image stacks that can be digitally re-sliced in any orientation for CHD<br />
assessment. Necropsy and EFIC imaging confirmed all RAA diagnoses (n=15/15), 77.4% <strong>of</strong> VSDs (n=72/93),<br />
and 91.2% <strong>of</strong> TGA/DORV/PA (n=31/34). We observed false positive rates <strong>of</strong> 17.2% (n=16/93) for VSDs and<br />
5.9% (n=2/34) for TGA/DORV, and a false negative rate <strong>of</strong> 5.4% (n=5.93) for VSDs. Overall, these findings<br />
indicate micro-CT is an invaluable tool for rapid screening and detection <strong>of</strong> a wide spectrum <strong>of</strong> CHD, the main<br />
limitation being the time required for image processing and diagnostic evaluation. Supported by U01-<br />
HL098180.<br />
1.18 Identification <strong>of</strong> Genetic Modifiers <strong>of</strong> Congenital Heart Defects<br />
Renita C. Polk and Roger H. Reeves<br />
Department <strong>of</strong> Physiology and The McKusick-Nathans Institute for Genetic Medicine, Johns Hopkins <strong>University</strong><br />
School <strong>of</strong> Medicine, Baltimore, MD 21205<br />
Congenital heart defects (CHD) are the most common congenital anomaly in live births. There is an especially<br />
high incidence <strong>of</strong> CHD in Down syndrome (DS), where 40 – 50% <strong>of</strong> affected individuals have a CHD. Thus,<br />
people with trisomy 21 are sensitized to CHD, but the fact that half <strong>of</strong> those with DS have a normal heart<br />
suggests that additional genetic and environmental factors interact with trisomy 21 to disrupt heart development.<br />
We have established a study to identify genetic risk factors for CHD in the sensitized DS population, genes<br />
whose individual contributions are too small to be identified on a euploid background (the DS Heart project,<br />
http://inertia.bs.jhmi.edu/ds/index.html). Here we used the Ts65Dn mouse model <strong>of</strong> DS as a corresponding<br />
sensitized population to study the role <strong>of</strong> the Tbx5 gene in CHD. Mutations <strong>of</strong> Tbx5 are associated with<br />
abnormalities <strong>of</strong> the heart and upper limbs. A Tbx5 null allele was crossed into Ts65Dn mice and newborn pups<br />
were sacrificed and examined for CHDs. We saw a significant difference between trisomic and euploid pups in<br />
the frequency <strong>of</strong> two particular defects, overriding aorta and an unusual connection between the right atrium and<br />
left ventricle. Almost 50% <strong>of</strong> the trisomic Tbx5 +/- mice present with overriding aorta and a ventricular septal<br />
defect, while less than 20% <strong>of</strong> the euploid pups have this defect. These results suggest that there is an<br />
interaction between Tbx5 and trisomy affecting heart development. Published studies with Tbx5 have<br />
suggested several genes as participants in this interaction. Future study will involve investigation into these<br />
gene(s) and the mechanisms behind this interaction.<br />
1.19 Confocal episcopic fluorescence image capture for phenotyping complex congenital heart defects<br />
and other structural malformations<br />
Richard JB Francis 1 , Wendy Shung 1 , Rod Bunn 2 , Andy Reidler 3 , Cecilia W. Lo* 1 .<br />
1 2<br />
Department <strong>of</strong> Developmental Biology, <strong>University</strong> <strong>of</strong> Pittsburgh School <strong>of</strong> Medicine, PA. Vashaw Scientific Inc,<br />
Norcross, GA, 3 Lecia Microsystems, Eldersburg, MD.<br />
We have previously shown episcopic fluorescence image capture (EFIC) is a powerful imaging tool for<br />
phenotyping complex structural heart defects. With EFIC imaging, paraffin embedded tissue is sectioned with a<br />
sledge microtome, and tissue aut<strong>of</strong>luorescence at the block face is captured by epifluorescence with each<br />
successive cut to generate registered 2D serial image stacks suitable for 2D digital reslicing or rapid 3D<br />
reconstructions. This allows complete diagnosis <strong>of</strong> even the most complex structural heart malformations. For<br />
standard EFIC imaging, light-blocking dye is added to the paraffin to prevent bleed through fluorescence from<br />
deeper tissue layers, a tedious and difficult process requiring superheating the wax. To overcome this<br />
requirement, we adapted EFIC imaging to use the Leica LSI confocal macroscope for confocal enhanced EFIC<br />
86
(CEFIC) imaging <strong>of</strong> standard paraffin embedded tissue. Using CEFIC, we characterized the congenital heart<br />
defects (CHD) in 435 fetal/newborn mice from 115 mutant mouse lines. A wide spectrum <strong>of</strong> CHD were<br />
observed, including ventricular septal defects (52%), ventricular hypertrophy (20.9%), ventricular noncompaction<br />
(17.5%), double outlet right ventricle (14.5%), overriding aorta (9%), transposition <strong>of</strong> the great<br />
arteries (3.7%), aortic stenosis (5.7%), pulmonary stenosis (4.1%), coronary fistulas (5.1%), major<br />
aortopulmonary collateral arteries (MAPCAs), interrupted aorta (2.5%), double aortic arches/vascular rings<br />
(2.1%), and hypoplastic left (0.7%) or right ventricles (0.9%). CEFIC can also identify other malformations<br />
difficult to ascertain by standard histology, such as tracheoesophageal fistulas and cloacal septation defects.<br />
We further showed CEFIC can be combined with fluorescence markers and also used for phenotyping other<br />
animals including the zebrafish.<br />
1.20 Mechanisms <strong>of</strong> Folate Protection <strong>of</strong> Cardiac Embryogenesis<br />
Mingda Han, Lifeng Zhang, and Kersti K. Linask<br />
USF Children’s Research Institute, Department <strong>of</strong> Pediatrics, USF Morsani College <strong>of</strong> Medicine, St. Petersburg,<br />
FL 33701<br />
We demonstrated that folate (FA) prevents cardiac birth defects acutely induced during gastrulation on<br />
embryonic day (ED) 6.75 by a binge level <strong>of</strong> alcohol, by the drug lithium (Li) or by homocysteine (HCy). Despite<br />
FA fortification, the mechanism <strong>of</strong> FA protection remains unknown. This study was to identify genes in the ED<br />
15.5 affected cardiac outflow tract (OFT) that are altered by maternal embryonic exposure to the above factors<br />
on ED 6.75, with and without FA supplementation. We used Affymetrix microarray analyses <strong>of</strong> gene expression<br />
changes in the ED 15.5 embryonic OFT tissues. Validation <strong>of</strong> specific gene expression was carried out on ED<br />
7.5 and again at ED 15.5, the latter in embryos displaying abnormal cardiac function as monitored by<br />
echocardiography. In the Li- affected OFT, 51 genes associated with Wnt and phosphatidylinositol signaling<br />
were down-regulated; 21 genes were up-regulated. Sixteen Wnt related genes were down-regulated by both Li<br />
and HCy; 23 were upregulated. A number <strong>of</strong> the genes overlapped. Thirty-eight genes that were misexpressed<br />
by Li and reversed by FA related to genes associated with chromatin modification reactions and with<br />
methylation. Key gene expression changes in the above pathways have been validated by RT-PCR and in situ<br />
hybridization. Taken together, these results confirm that Li, HCy and alcohol misexpress genes in Wnt/PtIn<br />
signaling and in FA metabolism that can lead to methylation and chromosome modification changes. Early<br />
supplementation with FA initiated right after conception protects one carbon metabolism, methylation reactions<br />
and gene expression leading to normal cardiogenesis.<br />
1.21 Value <strong>of</strong> high-throughput mutagenesis screening with fetal echocardiography for recovery <strong>of</strong> ENUinduced<br />
mouse mutations causing congenital heart disease<br />
Guozhen Chen 1 , Richard Francis 1 , Andrew Kim 1 , Wendy Shung 1 , Xiaoqin Liu 1 , Deborah Farkas 1 , Shane<br />
Anderton 1 , Heather Dawn Lynn 1 , Youngsil Kim 1 , Li Yin Wong 1 , Chien-Fu Chang 1 , Kimimasa Tobita 1 , Linda<br />
Leatherbury 2 , Cecilia W. Lo 1<br />
1<br />
Department <strong>of</strong> Developmental Biology, <strong>University</strong> <strong>of</strong> Pittsburgh School <strong>of</strong> Medicine, Pittsburgh, PA and<br />
2<br />
Children's National Medical Center, Washington, DC<br />
Compared to micro-computed tomography (micro-CT), episcopic fluorescence image capturing (EFIC),<br />
ultrasound has such disadvantage as lower spatial resolution, but with rapid, inexpensive and noninvasive, it is<br />
used for interrogating live pregnant mice <strong>of</strong> congenital heart disease (CHD). In the study, to improve the efficacy<br />
<strong>of</strong> ethylnitrosourea (ENU) mutagenesis high-throughput screening, we established a strategy <strong>of</strong> fetal<br />
echocardiography. First, fetal mice were rapidly screened using high frequency (15MHz) ultrasound (Acuson<br />
C512). Then, pregnant females with interesting fetuses were scanned carefully using ultra-high frequency<br />
(30~40MHz) ultrasound biomicroscopy (VEVO2100) with low penetration and limited width and depth range.<br />
With the strategy, 20,228 fetuses were screened between embryonic days 13.5 and 18.5 in utero, 1125(5.6%)<br />
showed developmental anomalies, with 683(60.7%) exhibiting cardiovascular defects, <strong>of</strong> which 125(24.4%) died<br />
prenatally at follow-up. There were 2915 fetuses further scanned carefully with ultrasound biomicroscopy,<br />
530(18.2%) showed developmental anomalies, with 390(73.6%) exhibiting a wide spectrum <strong>of</strong> CHD, including<br />
atrioventricular septal defects (15), conotruncal anomalies (43) (double outlet right ventricle, persistence truncus<br />
arteriosus, transposition <strong>of</strong> the great arteries, etc), laterality defects (20) (CHD with heterotaxy and situs<br />
inversus totalis), ectopiacordis (2), etc. In addition, growth retardation, hydrops, crani<strong>of</strong>acial/limb anomalies and<br />
body wall defects were highly associated with CHD. The results were confirmed by further anatomical analysis<br />
<strong>of</strong> Necropsy, micro-CT, EFIC in abnormal pups harvested immediately or euthanized after birth. In conclusion,<br />
ultrasound high-throughput screening with biomicroscopic scanning could play an essential role in<br />
87
cardiovascular phenotyping, and improve greatly the efficacy to recover ENU-induced mouse mutations causing<br />
CHD. (Supported by U01-HL098180)<br />
1.22 Identification <strong>of</strong> pathogenic copy number variations in syndromic CHDs by SNP array<br />
Zhi-Ping Tan 1, 2 , Jin-Lan Chen 1 , Can Huang 1 , Wei-Zhi Zhang 1, 2 , Jin-Fu Yang 1, 2 1, 2<br />
and Yi-Feng Yang<br />
1 2<br />
Department <strong>of</strong> Cardiothoracic Surgery, Clinical Center for Gene Diagnosis and Therapy <strong>of</strong> State Key<br />
Laboratory <strong>of</strong> Medical Genetics, The Second Xiangya Hospital, Central South <strong>University</strong>, Changsha, China<br />
High resolution SNP array has provided new opportunities to identify genetic lesions associated with human<br />
diseases such as congenital heart defects (CHDs). We screened a cohort <strong>of</strong> patients with syndromic congenital<br />
heart defects (CHD with extra-cardiac phenotypes) for pathogenic Copy Number Variations (pCNV) by SNParray<br />
and detected genomic lesions in 27 out <strong>of</strong> 180 (15%) patients. 78% <strong>of</strong> the affected individuals (21/27)<br />
have an aberrant chromosomal region containing a gene previously known to be associated with CHDs. Six<br />
patients had novel CNVs that contain candidate genes involved in cardiogenesis. Specially, two CNVs with the<br />
3p26.1-26.3del/10p15.3-15.1dup were found in a heterozygous twin with complex CHD. Interestingly, a<br />
candidate gene for ventricular aneurysms and septal defects was recently mapped in the genomic region 10p15<br />
[Tremblay et al, Eur Heart J. 2011, 32 (5):568-73], which overlaps with the genomic region containing<br />
the10p15.3-15.1dup. Further analysis <strong>of</strong> these CNVs and candidate genes identified in these CNVs will be<br />
presented.<br />
1.23 Mutations in the T (brachyury) gene cause a novel syndrome consisting <strong>of</strong> sacral agenesia,<br />
defective ossification <strong>of</strong> the vertebral bodies and persistent notochord<br />
AV Postma 1 , M Alders 2 , M Sylva 1 , CM Bilardo 3,4 , E Pajkrt 3 , RR van Rijn 5 , A Ilgun 1 , P Barnett 1 , MMAM Mannens 2 ,<br />
AFM Moorman 1 , RJ Oostra 1 , MC van Maarle 2<br />
1<br />
Heart Failure Research Centre, Dept <strong>of</strong> Anatomy and Embryology, Academic Medical Centre, Amsterdam, The<br />
Netherlands<br />
2<br />
Dept <strong>of</strong> Clinical Genetics, Academic Medical Centre, Amsterdam, The Netherlands<br />
3<br />
Dept <strong>of</strong> Obstetrics and Gynaecology, Academic Medical Centre, Amsterdam, The Netherlands<br />
4<br />
Dept <strong>of</strong> Obstetrics and Gynaecology, <strong>University</strong> Medical Centre, Groningen, The Netherlands<br />
5<br />
Dept <strong>of</strong> Radiology, Academic Medical Centre, Amsterdam, The Netherlands<br />
The T gene (Brachyury gene) is the founding member <strong>of</strong> the T-box family <strong>of</strong> transcription factors. The protein<br />
encoded by this gene is an embryonic nuclear transcription factor that binds to specific DNA elements and<br />
effects transcription <strong>of</strong> genes required for mesoderm formation and differentiation. The protein is localized to<br />
notochord-derived cells and is vital for the formation and differentiation <strong>of</strong> the posterior mesoderm and axial<br />
development <strong>of</strong> all vertebrates. We report here on four patients from three consanguineous families exhibiting<br />
sacral agenesia, delayed ossification <strong>of</strong> the vertebral bodies and a persistent notochord, the heart was<br />
unaffected. Homozygosity mapping identified a common 4.1Mb homozygous region on chromosome 6q27,<br />
containing the T gene. Sequencing <strong>of</strong> T in the affected individuals led to the identification <strong>of</strong> a homozygous<br />
mutation, p.H171R, in the highly conserved T-box and absent from control alleles. The homozygous mutation<br />
results in diminished DNA binding, interferes with bone morphogenetic protein (BMP) signaling and causes<br />
changes in expression <strong>of</strong> genes involved in notochord maintenance and chondrogenesis. In conclusion, we<br />
present evidence that a novel syndrome consisting <strong>of</strong> sacral agenesia, defective ossification <strong>of</strong> the vertebral<br />
bodies and persistent notochord is caused by a homozygous mutation in the T gene. The mutation leads to a<br />
moderate loss <strong>of</strong> function on DNA binding and disturbed expression <strong>of</strong> important downstream targets genes<br />
involved in proper posterior mesoderm and axial development, likely resulting in the unique phenotype.<br />
1.24 TWO COMPOUND HETEROZYGOUS MUTATIONS in NFATC1 in a LEBANESE PATIENT with<br />
TRICUSPID ATRESIA<br />
Zahy AbdulSater 1 , Amin Yehya 1 , Jean Beresian 1 , Elie Salem 1 , Amina Kamar 1 , Serine Baydoun 1 , Kamel<br />
Shibbani 1 , Ayman Soubra 1 , Fadi Bitar 2,* , and Georges Nemer 1,*<br />
1 2<br />
Departments <strong>of</strong> Biochemistry and Molecular Genetics, Pediatrics and Adolescent Medicine, American<br />
<strong>University</strong> <strong>of</strong> Beirut, Beirut, Lebanon<br />
Tricuspid Atresia (TA) is a rare form <strong>of</strong> congenital heart disease (CHD) with usually poor prognosis in humans. It<br />
presents as a complete absence <strong>of</strong> the right atrio-ventricular connection secured normally by the tricuspid valve.<br />
Defects in the tricuspid valve are so far not associated with any genetic locus, although mutations in numerous<br />
genes were linked to multiple forms <strong>of</strong> congenital heart disease. In the last decade, Knock-out mice have<br />
88
<strong>of</strong>fered models for cardiologists and geneticists to study the causes <strong>of</strong> congenital disease. One such model was<br />
the Nfatc1-/- mice embryos which die at mid-gestation stage due to a complete absence <strong>of</strong> the valves. NFATC1<br />
belongs to the Rel family <strong>of</strong> transcription factors members <strong>of</strong> which were shown to be implicated in gene<br />
activation, cell differentiation, and organogenesis. We have previously shown that a tandem repeat in the<br />
intronic region <strong>of</strong> NFATC1 is associated with ventricular septal defects. In this report, we unravel for the first<br />
time a direct link between a mutation in NFATC1 and TA. Two heterozygous missense mutations were found in<br />
the NFATC1 gene in one indexed-case out <strong>of</strong> 19 patients with TA. The two amino-acids changes were not found<br />
in other patients with CHDs nor in the control healthy population. Moreover, we showed that these mutations<br />
alter dramatically the normal function <strong>of</strong> the protein at the localization, DNA binding and transcriptional levels<br />
suggesting they are disease-causing.<br />
1.25 Actin Mutation that Causes Patent Ductus Arteriosus Alters Regulation by Formin<br />
Kuo-Kuang Wen 1 , Lindsey E. Malloy 2 , Alyson R. Pierick 2 , Elesa W. Wedemeyer 2 , Sarah E. Bergeron 1,2 , Nicole<br />
D. Vanderpool 1 , Melissa McKane 1 , Peter A. Rubenstein 1 and Heather L. Bartlett 1,2<br />
Department <strong>of</strong> Biochemistry 1 and Department <strong>of</strong> Pediatrics 2 , Roy A. and Lucille A. Carver College <strong>of</strong> Medicine,<br />
<strong>University</strong> <strong>of</strong> Iowa, Iowa City, Iowa 52242<br />
More than thirty mutations in ACTA2, which encodes α-smooth muscle actin, have been identified to cause<br />
autosomal dominant cardiovascular disease. All mutations cause thoracic aortic aneurysm but the R256H<br />
mutation is <strong>of</strong> interest because it distinctively causes patent ductus arteriosus. R256H is one <strong>of</strong> the more<br />
prevalent mutations and, based on its molecular location near the strand-strand interface in the actin filament,<br />
may affect F-actin stability. To understand the molecular ramifications <strong>of</strong> the R256H mutation, we generated<br />
Saccharomyces cerevisiae yeast cells expressing only R256H yeast actin as a model system. These cells<br />
displayed abnormal cytoskeletal morphology and increased sensitivity to Latrunculin A. After cable disassembly<br />
induced by transient exposure to Latrunculin-A, mutant cells were delayed in reestablishing the actin<br />
cytoskeleton. In vitro, mutant actin exhibited a higher than normal critical concentration and a delayed<br />
nucleation. Consequently, we investigated regulation <strong>of</strong> mutant actin by formin, a potent facilitator <strong>of</strong> nucleation<br />
and a protein needed for normal vascular smooth muscle cell development. Mutant actin polymerization was<br />
inhibited by the FH1-FH2 fragment <strong>of</strong> the yeast formin, Bni1. This fragment strongly capped the filament rather<br />
than facilitating polymerization. Interestingly, phalloidin or the presence <strong>of</strong> wild type actin reversed the strong<br />
capping behavior <strong>of</strong> Bni1. Together, the data suggest that the R256H actin mutation alters filament conformation<br />
resulting in instability and misregulation by formin. These biochemical effects may contribute to inappropriate<br />
persistence <strong>of</strong> the ductus arteriosus.<br />
1.26 Perturbation <strong>of</strong> the titin/MURF1 signaling complex is associated with hypertrophic cardiomyopathy<br />
in a fish model and in human patients<br />
Shinji Makino, 1,4 Yuta Higashikuse, 1,2 Takuro Arimura, 3 Sung Han Yoon, 1 Toshimi Kageyama, 1 Mayumi Oda, 1,4<br />
Hirokazu Enomoto, 1 Motoaki Sano, 1 Shinsuke Yuasa, 1,4 Ruri Kaneda, 1 Mitsushige Murata, 1 Fumiyuki Hattori, 1<br />
Takeshi Suzuki, 2 Atsushi Kawakami, 5 Alexander Gasch, 7 Tetsushi Furukawa, 8 Akira Kudo, 5 Siegfried Labeit, 7<br />
Akinori Kimura 3,6 and Keiichi Fukuda 1<br />
1 Cardiology Division, Department <strong>of</strong> Internal Medicine, Keio <strong>University</strong> School <strong>of</strong> Medicine, Tokyo, Japan.<br />
2 Division <strong>of</strong> Basic Biological Sciences, Faculty <strong>of</strong> Pharmacy, Keio <strong>University</strong>, Tokyo, Japan. 3 Department <strong>of</strong><br />
Molecular Pathogenesis, Medical Research Institute, Tokyo Medical and Dental <strong>University</strong>, Tokyo, Japan.<br />
4 Center for Integrated Medical Research, Keio <strong>University</strong> School <strong>of</strong> Medicine, Tokyo, Japan. 5 Department <strong>of</strong><br />
Biological Information, Tokyo Institute <strong>of</strong> Technology, Yokohama, Japan. 6 Laboratory <strong>of</strong> Genome Diversity,<br />
Graduate School <strong>of</strong> Biomedical Science, Tokyo Medical and Dental <strong>University</strong>, Tokyo, Japan. 7 Department <strong>of</strong><br />
Integrative Pathophysiology, Medical Faculty Mannheim, Mannheim, Germany 8 Department <strong>of</strong> Bio-informational<br />
Pharmacology, Medical Research Institute, Tokyo Medical and Dental <strong>University</strong>, Japan.<br />
Background: Hypertrophic cardiomyopathy (HCM) is a hereditary disease characterized by cardiac hypertrophy<br />
with diastolic dysfunction. Gene mutations causing HCM have been found in about half <strong>of</strong> the patients, while<br />
pathogenesis remain unknown for many cases <strong>of</strong> HCM. To identify novel mechanisms underlying HCM<br />
pathogenesis, we performed forward-genetic N-ethyl-N-nitrosourea (ENU) mutagenesis screening. Methods<br />
and Results: We generated a cardiovascular-mutant medaka fish non-spring heart (nsh), which showed diastolic<br />
dysfunction and hypertrophic myocardium. The nsh mutant heart lost elasticity and beat in a rigid manner, as<br />
determined by high-speed video imaging with the analysis using motion vector prediction (MVP) method. The<br />
nsh homozygotes had disrupted sarcomeres and expressed pathologically stiffer titin is<strong>of</strong>orms. In addition, the<br />
nsh heterozygotes showed M-line disassembly that is similar to the pathological changes found in HCM. By<br />
89
positional cloning, we identified a missense mutation in an immunoglobulin (Ig) domain located in the M-line-Aband<br />
transition zone <strong>of</strong> titin, leading to D23186V around the binding site for muscle-specific ring finger protein 1<br />
(MURF1). Analysis <strong>of</strong> 96 genetically unrelated Japanese patients with familial HCM, who had no previously<br />
implicated mutations in known sarcomeric gene candidates, identified two disease-associated mutations in the<br />
other Ig domains in proximity to the M-line region <strong>of</strong> titin. In vitro studies revealed that the mutations found in<br />
both medaka fish and in familial HCM increased binding <strong>of</strong> titin to MURF1 and enhanced titin degradation by<br />
ubiquitination. Conclusions: These findings implicate an impaired interaction between titin and MURF1 as a<br />
novel mechanism underlying the pathogenesis <strong>of</strong> HCM.<br />
Section 2: Cardiomyocyte Growth and Regeneration<br />
S2.1 Cardiac hypertrophy in LEOPARD Syndrome is caused by PTPN11 loss-<strong>of</strong>-function activity in the<br />
developing endocardium<br />
Jessica Lauriol 1 , Kimberly Keith 1 , Boding Zhang 3 , Roderick Bronson 2 , Kyu-Ho Lee 3 , and Maria I. Kontaridis 1,4<br />
1 Department <strong>of</strong> Medicine, Division <strong>of</strong> Cardiology, Beth Israel Deaconess Medical Center, Boston, MA.<br />
2 Department <strong>of</strong> Pathology, Harvard Medical School, Boston, MA.<br />
3 Department <strong>of</strong> Cardiovascular Developmental Biology, Medical <strong>University</strong> <strong>of</strong> South Carolina, Charleston, SC.<br />
4 Department <strong>of</strong> Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA.<br />
Mutations in PTPN11, encoding the protein tyrosine phosphatase (PTP) SHP2, cause LEOPARD Syndrome<br />
(LS), an autosomal dominant disorder with multiple cardiac defects, including hypertrophy. Interestingly, LS<br />
mutations are catalytically inactive and behave as loss-<strong>of</strong>-function. However, how LS mutants affect cardiac<br />
development remains unclear. We generated an inducible “knockin” mouse model expressing the LSassociated<br />
Ptpn11 Y279C mutation (iLS/+). When crossed to EIIA deleter-Cre mice, these (now termed) LS/+<br />
mice recapitulated nearly all aspects <strong>of</strong> the human LS phenotype, including hypertrophy. To determine whether<br />
defects in LS/+ mice could be attributed directly to aberrant cardiac developmental effects, we examined hearts<br />
from WT, LS/+ and LS/LS embryos at E10.5, E14.5, and E15.5. Both LS/+ and LS/LS developing hearts had<br />
significantly diminished trabeculation at E10.5, as well as valvular hyperplasia and ventricular septal defects<br />
(VSD) at E14.5, indicative <strong>of</strong> defective or delayed cardiac development. In addition, LS/LS embryos had<br />
persistent VSD at E15.5 and cardiac looping defects that resulted in dextraposition <strong>of</strong> the aorta, which led to<br />
embryonic lethality between E14.5 and E15.5. To determine the lineage-specific effects, we generated neural<br />
crest (Wnt1::Cre)-, endothelial (Tie2::Cre)-, and myocardial (Nkx2.5::Cre)- specific LS/+ expressing mouse lines.<br />
Surprisingly, we found that only the endocardial-specific LS-expressing mice could completely recapitulate the<br />
embryonic cardiac developmental defects observed in the LS/+ phenotype. Moreover, these mice also<br />
developed the adult-onset hypertrophy, as observed in adult LS/+ mice. Taken together, our data indicate that<br />
the adult-onset cardiac hypertrophy associated with LS is caused by PTPN11 loss-<strong>of</strong>-function effects that occur<br />
in the developing endocardium.<br />
S2.2 Epigenetic regulation <strong>of</strong> cardiac hypertrophy<br />
Qing-Jun Zhang 1 , David Maloney 2 , Anton Simeonov 2 , and Zhi-Ping Liu 1<br />
1 Department <strong>of</strong> Internal medicine, division <strong>of</strong> Cardiology, UT Southwestern<br />
2 National Institutes <strong>of</strong> Health Chemical Genomics Center, National Human Genome Research Institute.<br />
One <strong>of</strong> the major challenges in managing and treating heart failure patients is to develop disease-modifying<br />
drugs that can prevent, reverse, or slow down the disease progression. Upon pathological insults, the heart<br />
undergoes remodeling processes, including left ventricular hypertrophy and reprogramming <strong>of</strong> gene expression.<br />
Understanding the mechanisms involved could provide a key to develop interventional therapeutics. Epigenetic<br />
modification <strong>of</strong> chromatin, including histone methylation, regulates gene transcription in response to<br />
environmental signals. JMJD2A is a trimethyl-lysine specific histone lysine demethylase. To study the role <strong>of</strong><br />
JMJD2A, we generated heart specific JMJD2A overexpression and deletion mouse lines. Our studies with these<br />
genetically modified mice indicated that JMJD2A is required for pathological cardiac hypertrophy. Furthermore,<br />
we show that the demethylase activity <strong>of</strong> JMJD2A is required for its transcriptional activity. To test whether<br />
inhibition <strong>of</strong> JMJD2A enzymatic activity suppresses hypertrophic response, we identified several small molecule<br />
inhibitors <strong>of</strong> JMJD2A. These small molecule inhibitors <strong>of</strong> JMJD2A inhibited the phenylephrine-stimulated<br />
cardiomyocyte hypertrophy in vitro. Our data suggests that JMJD2A enzymatic activity may act as a<br />
hypertrophic determinant and may be an innovative drug target for prevention and treatment <strong>of</strong> pathological<br />
cardiac hypertrophy and heart failure.<br />
90
S2.3 Regulation <strong>of</strong> striated muscle gene expression: A role for Prox1 during cardiac and skeletal<br />
muscle development<br />
Louisa K. Petchey 1 , Paul R. Riley 1<br />
1 Molecular Medicine Unit, UCL Institute <strong>of</strong> Child Health, London WC1N 1EH, UK<br />
Is<strong>of</strong>orm-specific expression <strong>of</strong> muscle structural genes is a crucial component <strong>of</strong> the structural, functional and<br />
metabolic distinction that exists between the fibre types comprising striated cardiac and skeletal muscle. The<br />
homeobox transcription factor Prox1 has previously been shown to be expressed in slow-twitch skeletal muscle<br />
in zebrafish and to be required for normal cardiac muscle development in mice. Using Nkx2.5-Cre, Myf5-Cre<br />
and Prox1 flox mouse lines to knockout Prox1 in both striated muscle types, we can identify for the first time a role<br />
for Prox1 in the repression <strong>of</strong> three key fast-twitch skeletal muscle genes, Tnnt3, Tnni2 and Myl1, in both<br />
cardiac and slow-twitch skeletal muscle. Prox1 has an additional role in skeletal muscle in the regulation <strong>of</strong><br />
myosin heavy chain is<strong>of</strong>orm expression. This implicates Prox1 in a complex regulatory network that determines<br />
fibre type in skeletal muscle, where it functions downstream <strong>of</strong> Sox6 and upstream <strong>of</strong> Six1; and characterises a<br />
novel role for Prox1 during heart development. The inappropriate expression <strong>of</strong> fast-twitch skeletal muscle<br />
genes is known to negatively impact cardiac function while some human cardiac and skeletal myopathies have<br />
been associated with incorrect is<strong>of</strong>orm expression <strong>of</strong> structural genes. New opportunities for intervening in the<br />
molecular mechanisms that underlie these pathologies will be gained from a better understanding <strong>of</strong> their<br />
developmental regulation.<br />
S2.4 CIP, a novel cardiac nuclear protein regulates cardiac function and remodeling<br />
Zhan-Peng Huang, Hee Young Seok, Jinghai Chen, and Da-Zhi Wang<br />
Department <strong>of</strong> Cardiology Children's Hospital Boston Harvard Medical School 320 Longwood Avenue Boston,<br />
MA 02115<br />
Mammalian heart has minimal regenerative capacity. In response to mechanical or pathological stress, the heart<br />
undergoes cardiac remodeling. Pressure and volume overload in the heart cause increased size (hypertrophic<br />
growth) <strong>of</strong> cardiomyocytes. Whereas the regulatory pathways that activate cardiac hypertrophy have been well<br />
established, the molecular events that inhibit or repress cardiac hypertrophy are less known. Here, we report the<br />
identification, characterization and functional examination <strong>of</strong> CIP, a novel cardiac Isl1-interacting protein. CIP<br />
was identified from a bioinformatic search for novel cardiac-expressed genes in mouse embryonic hearts. CIP<br />
encodes a nuclear protein without recognizable motifs. Northern blotting, in situ hybridization and reporter gene<br />
tracing demonstrated that CIP is highly expressed in cardiomyocytes <strong>of</strong> developing and adult hearts. Yeast-twohybrid<br />
screening identified Isl1, a LIM/homeodomain transcription factor essential for the specification <strong>of</strong> cardiac<br />
progenitor cells in the second heart field, as a co-factor <strong>of</strong> CIP. CIP directly interacted with Isl1 and we mapped<br />
the domains <strong>of</strong> these two proteins which mediate their interaction. We show that CIP represses the<br />
transcriptional activity <strong>of</strong> Isl1 in the activation <strong>of</strong> the MEF2C enhancer. The expression <strong>of</strong> CIP was dramatically<br />
reduced in hypertrophic cardiomyocytes. Most importantly, overexpression <strong>of</strong> CIP repressed agonist-induced<br />
cardiomyocyte hypertrophy. Interestingly, recent work showed that CIP also interacts with Lamin A/C (LMNA), a<br />
causative gene mutated in patients with DCM and other disorders. We found that the expression <strong>of</strong> CIP was<br />
markedly regulated in animal models <strong>of</strong> cardiac diseases, including HCM and DCM. Furthermore, our<br />
preliminary data showed that CIP knockout mice display impaired cardiac function. Together, our studies identify<br />
CIP a novel regulator <strong>of</strong> cardiac remodeling and disease.<br />
S2.5 Yap1 regulates cardiomyocyte proliferation through PIK3CB<br />
Zhiqiang Lin(a), Alexander von Gisea(a), Pingzhu Zhou(a), Jessie Buck(a), William Pu(a,b)<br />
(a)Department <strong>of</strong> Cardiology and (b)Stem Cell Program, Children’s Hospital Boston, Boston, MA 02115<br />
Cardiomyocyte loss is a major underlying cause <strong>of</strong> heart failure. Works focusing on cardiomyocyte proliferation<br />
regulation have shown that adult cardiomyocytes do proliferate to a limited extent, but not enough to show any<br />
observable clinical benefits. Efforts are needed to deciphering genes capable <strong>of</strong> driving cardiomyocytes to reenter<br />
cell cycle. The Hippo kinase cascade is an important regulator <strong>of</strong> organ growth, including heart. A major<br />
target <strong>of</strong> this kinase cascade is YAP1. Our data showed that fetal Yap1 inactivation caused marked, lethal<br />
myocardial hypoplasia and decreased cardiomyocyte proliferation, whereas fetal activation <strong>of</strong> YAP1 stimulated<br />
cardiomyocyte proliferation. Remarkably, YAP1 activation was sufficient to stimulate proliferation <strong>of</strong> postnatal<br />
cardiomyocytes, both in culture and in the intact heart. To characterize the mechanism <strong>of</strong> YAP1 induced<br />
cardiomyocyte proliferation, we carried out microarray and Chip sequencing assays. By combining the<br />
microarray da ta and the Chip sequencing results, we found several genes that possibly regulated by YAP1.<br />
91
PIK3CB was validated to be a direct target <strong>of</strong> Yap1, and a regulatory element residing in the first intron <strong>of</strong><br />
PIK3CB was characterized. Knocking down <strong>of</strong> PIK3CB significantly reduced the YAP1 induced cardiomyocyte<br />
proliferation, however, TGX221, a specific PIK3CB kinase inhibitor failed to weaken the effects <strong>of</strong> YAP1 on<br />
cardiomyocyte proliferation. Overexpression <strong>of</strong> either PIK3CB, or PIK3CBK805R, a kinase dead mutation<br />
version, was sufficient to drive neonatal cardiomyocytes to re-enter cell cycle in vitro. These studies<br />
demonstrate that YAP1 is a crucial regulator <strong>of</strong> cardiomyocyte proliferation, and YAP1 regulates cardiomyocyte<br />
proliferation partially through PIK3CB.<br />
S2.6 A Transitional Extracellular Matrix Instructs Epicardial-mediated Vertebrate Heart Regeneration<br />
Sarah Mercer 1 , Claudia Guzman 1 , Chia-ho Cheng 2 , Shannon Odelberg 3 , Ken Marx 2 and Hans-Georg Simon 1<br />
1<br />
Department <strong>of</strong> Pediatrics and Children’s Memorial Research Center, Northwestern <strong>University</strong> Feinberg School<br />
<strong>of</strong> Medicine, <strong>Chicago</strong> IL, USA<br />
2<br />
Department <strong>of</strong> Chemistry, <strong>University</strong> <strong>of</strong> Massachusetts-Lowell, USA<br />
3<br />
Department <strong>of</strong> Internal Medicine, <strong>University</strong> <strong>of</strong> Utah School <strong>of</strong> Medicine, Salt Lake City UT, USA<br />
Unlike humans, certain vertebrates including newts and zebrafish possess extraordinary abilities to functionally<br />
regenerate lost appendages and injured organs, including cardiac muscle. We aim to discover the underpinning<br />
mechanisms that regulate complex tissue rebuilding in these regeneration-competent species, ultimately<br />
developing strategies for regenerative wound healing in mammals. Here, we present new evidence that the<br />
extracellular matrix (ECM) provides signals to tissues and cells essential for the induction and maintenance <strong>of</strong><br />
regenerative processes in cardiac muscle. Comprehensive mining <strong>of</strong> DNA microarrays <strong>of</strong> regenerating newt and<br />
zebrafish hearts with data comparison to the scar-forming response following myocardial infarction in mice and<br />
humans provided a signature <strong>of</strong> conserved regenerative gene activities. Differential expression and Gene<br />
Ontology analyses revealed that distinct ECM components and ECM-modifying proteases are among the<br />
earliest upregulated and most significantly enriched genes in response to injury in both the newt and zebrafish.<br />
In contrast, following mammalian cardiac injury, immune and inflammatory responses are significantly activated<br />
instead <strong>of</strong> ECM genes. Complementary immunohistochemistry studies in the newt demonstrated dynamic<br />
spatial and temporal changes in ECM composition between normal and regenerating heart tissues, especially in<br />
the epicardium early in the regenerative response. We show in vivo and under defined culture conditions that<br />
the proliferative and migratory response <strong>of</strong> cardiomyocytes is directly linked to distinct matrix remodeling in both<br />
the myocardium and epicardium <strong>of</strong> the regenerating newt heart. Collectively, these results provide a novel<br />
understanding <strong>of</strong> the regenerative process, suggesting that an evolutionarily conserved, regeneration-specific<br />
matrix instructs cell activities essential for cardiac muscle regeneration.<br />
2.7 Cardiomyocyte proliferation contributes to post-natal heart growth in humans<br />
Kevin Bersell 1 , Mariya Mollova 1 , Stuart Walsh 1 , Jainy Savla 1 , Tanmoy DasLala 1 , Shin-Young Park 1 , Leslie<br />
Silberstein 1 , Cris dosRemedios 2 , Dionne Graham 1 , Steven Colan 1 and Bernhard Kühn 1 .<br />
1 Children's Hospital Boston, 2 <strong>University</strong> <strong>of</strong> Sydney<br />
Cardiomyocyte proliferation is an active cellular mechanism during myocardial growth and regeneration in<br />
zebrafish, newt, and neonatal mice. We hypothesized that cardiomyocyte proliferation may also contribute to the<br />
growth <strong>of</strong> the human myocardium between birth and adulthood. To test this hypothesis, we analyzed the<br />
cellular growth mechanisms in a set <strong>of</strong> 20 human hearts (age 3 weeks – 20 years) that were procured for<br />
transplantation and free from myocardial diseases. Measurements <strong>of</strong> mitosis, made by automated<br />
quantification <strong>of</strong> phosphorylated histone-3-positive cardiomyocytes, showed that during the first year <strong>of</strong> life the<br />
mean percentage <strong>of</strong> cardiomyocytes undergoing mitosis was 0.05%, declining to 0.01% at 20 years <strong>of</strong> age.<br />
Cardiomyocyte cytokinesis, visualized by an antibody against MKLP-1 (a component <strong>of</strong> the centralspindlin<br />
complex), was detectable up to 20 years <strong>of</strong> age, but not later in life. The number <strong>of</strong> cardiomyocytes, quantified<br />
with stereology, increased four-fold between birth and 20 years. The mean cardiomyocyte volume increased<br />
concomitantly 12-fold. Relating these mechanistic data to left ventricular mass (determined by<br />
echocardiography) showed that cardiomyocyte proliferation contributes 37% to physiologic myocardial growth<br />
between birth and 20 years. These findings suggest that altered cardiomyocyte proliferation may be involved in<br />
abnormal myocardial growth in congenital heart disease. Our findings also suggest that myocardial regeneration<br />
may be present or stimulated in young humans since the underlying mechanism, cardiomyocyte proliferation, is<br />
active under the age <strong>of</strong> 20 years.<br />
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2.8 Microarray and Ultrastructure Analyses <strong>of</strong> a Regenerative Myocardium<br />
Pooja Pardhanani 1 , Jeremy Barth 2 , Heather J. Evans-Anderson 1<br />
1 Winthrop <strong>University</strong>, Rock Hill, SC 29733<br />
2 Medical <strong>University</strong> <strong>of</strong> South Carolina, Charleston, SC 29425<br />
Ciona intestinalis is an invertebrate animal model system that is well characterized and has many advantages<br />
for the study <strong>of</strong> cardiovascular biology. A striking difference between most vertebrates and Ciona is that the<br />
Ciona myocardium is capable <strong>of</strong> regenerating cardiac myocytes throughout its lifespan, which makes the<br />
regulatory mechanisms <strong>of</strong> cardiac myocyte proliferation in Ciona intriguing. In order to identify important<br />
regeneration factors in Ciona, microarray analysis was conducted on RNA from adult Ciona hearts with normal<br />
or damaged myocardium using custom Affymetrix GeneChips. Hearts were injured via ligation or cryoinjury to<br />
stimulate regeneration. After a 24 or 48 hour recovery period, total RNA was isolated from damaged and control<br />
hearts. Initial results indicate significant changes in gene expression in hearts damaged by ligation in<br />
comparison to cryoinjured or control hearts. Ligation injury shows differential expression <strong>of</strong> 223 genes as<br />
compared to control (fold change >2, p
positive cardiomyocytes, epicardium, and endocardium, at various times after two different models <strong>of</strong> cardiac<br />
injury. Through translational pr<strong>of</strong>iling <strong>of</strong> cardiomyocytes, we have begun to identify new injury responses by<br />
cardiomyocytes that are critical for heart regeneration in zebrafish.<br />
2.11 Rheb is essential for post-natal heart function<br />
Yunshan Cao 1,2 , Xiangqi Wu 1,2 Xinli Li 2 , Zhongzhou Yang 1<br />
1 Ministry <strong>of</strong> Education Key Laboratory <strong>of</strong> Model Animal for Disease Study, Model Animal<br />
Research Centre, Nanjing <strong>University</strong>, Nanjing, China<br />
2 Department <strong>of</strong> Cardiology, the First Affiliated Hospital <strong>of</strong> Nanjing Medical <strong>University</strong>, Nanjing, China<br />
Rheb (Ras homologue enriched in brain) activates mTOR complex 1 (mTORC1) and is an energy sensor. Rheb<br />
plays a critical role to keep the balance between cell growth and stress response through regulating protein<br />
synthesis and degradation. In this study, we attempted to clarify the role <strong>of</strong> Rheb in the post-natal heart through<br />
studying cardiomyocyte-specific Rheb-deficient mice. Cardiomyocyte-specific Rheb deletion mice were born in<br />
Mendelian ratio but started to die 12 days after birth. All <strong>of</strong> them were lost by 16 days after birth.<br />
Echocardiographic analysis revealed that Rheb-deficient mice exhibited cardiac dilatation and reduced<br />
contractility 12 days after birth. Electrocardiography recordings showed intermittent complete heart block and<br />
sinus arrest in the mutant. These suggest that Rheb-deficient mice died <strong>of</strong> heart failure and arrhythmia. Rhebdeletion<br />
mice also showed reduced heart weight and cardiac fiber size. Downstream <strong>of</strong> mTORC1, S6 and<br />
4EBP1 were shown to have drama tically reduced phosphorylation levels with enhanced Akt activity. Thus, we<br />
concluded that Rheb-mTOR pathway in the heart is essential to regulate heart contractility and<br />
electrophysiologic homeostasis at young age.<br />
2.12 Wdr1 is essential for post-natal and adult heart function<br />
Baiyin Yuan, Zhongzhou Yang<br />
Model Animal Research Center, Nanjing <strong>University</strong><br />
WDR1, a WD-repeat protein, is highly conserved across all species, from yeast and plants to mammals. it is<br />
considered as an important c<strong>of</strong>actor <strong>of</strong> c<strong>of</strong>ilin, which serves to accelerate c<strong>of</strong>ilin’s activity. AIP1 alone has<br />
negligible effects on actin filament dynamics, whereas in the presence <strong>of</strong> ADF/c<strong>of</strong>ilin, AIP1 enhances filament<br />
fragmentation by capping ends <strong>of</strong> severed filaments. So far, most in vivo analyses <strong>of</strong> AIP1 come from C.<br />
elegans and few unicellular organisms. unc-78, an AIP1 gene in the C. elegans, is required for organized<br />
assembly <strong>of</strong> sarcomeric actin filaments in the body wall muscle. However, the function <strong>of</strong> WDR1 in vertebrate<br />
muscle has not been reported. Here we generated a WDR1 conditional knockout mouse, aMHC promoter<br />
drivened cardiac-specific deletion WDR1 in heart caused mice dead from post-natal day 13 to day 24 ,<br />
accompanied by the increasing <strong>of</strong> total actin and hypertrophy; to test whether Wdr1 was essential for adult<br />
heart, Tamoxifen induced cardiomyocyte-specific deletion <strong>of</strong> Wdr1 in adult mice, This ablation resulted in the<br />
death within 2 months <strong>of</strong> Wdr1 deletion. These studies revealed an essential requirement for Wdr1 in post-natal<br />
and adult heart functions.<br />
2.13 Preservation <strong>of</strong> Muscle Force in Mdx3cv Mice Correlates with the Low-level Expression <strong>of</strong> a Near<br />
Full-length Dystrophin Protein<br />
Li, Dejia; Yue, Yongping; Duan, Dongsheng<br />
Univ. <strong>of</strong> Missouri, Columbia, MO 65212<br />
Complete absence <strong>of</strong> dystrophin causes Duchenne muscular dystrophy (DMD). Dystrophin restoration at ≥ 20%<br />
level reduces muscle pathology and improves muscle force. Levels lower than this are considered<br />
therapeutically irrelevant. Interestingly, less than 20% dystrophin expression is seen in some Becker muscular<br />
dystrophy (BMD) patients. To understand the role <strong>of</strong> low-level dystrophin expression, we compared muscle<br />
force and pathology in mdx3cv and mdx4cv mice. Dystrophin was eliminated in mdx4cv mice. But mdx3cv mice<br />
expressed a near full-length dystrophin protein at ~5% <strong>of</strong> the normal level. Consistent with previous reports, we<br />
found dystrophic skeletal muscle pathology in both strains. Surprisingly, mdx3cv extensor digitorium longus<br />
(EDL) muscle showed significantly higher tetanic force and it was also more resistant to eccentric contractioninduced<br />
injury. Furthermore, mdx3cv forelimb grip force was stronger. Immunostaining revealed utrophin upregulat<br />
ion and detectable d ystrophin-associated glycoprotein complex assembly on the sarcolemma in both<br />
strains. Our results suggest that a sub-therapeutic level expression <strong>of</strong> a near full-length membrane-bound<br />
dystrophin may have contributed to muscle force preservation in mdx3cv mice. This finding may help to explain<br />
the benign clinical phenotype in some BMD patients. (Supported by NIH and MDA).<br />
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2.14 Conditional Ablation <strong>of</strong> Ezh2 in Murine Hearts Reveals Its Essential Roles in Endocardial Cushion<br />
Formation, Cardiomyocyte Proliferation and Survival<br />
Li Chen 1 , Yanlin Ma 3 , Wei Yu 2 , Robert J Schwartz 1-2 , Ling Qian 1 , Jun Wang 1<br />
1 Texas Heart Institute, Houston, TX 77030, USA. 2 <strong>University</strong> <strong>of</strong> Houston, Houston, TX 77204, USA. 3 Institute <strong>of</strong><br />
Biosciences and Technology, Texas A&M Health Science Center, Houston, TX, 77030 USA.<br />
Ezh2 is a histone trimethyltransferase that mainly silences genes via catalyzing trimethylation <strong>of</strong> histone 3 lysine<br />
27 (H3K27Me3). The role <strong>of</strong> Ezh2 as a regulator <strong>of</strong> gene silencing and cell proliferation in cancer development<br />
has been investigated; however, its function in heart development during embryonic cardiogenesis has not been<br />
well studied. We conditionally ablated Ezh2 in murine heart using the conditional Ezh2 allele and Nkx2.5-cre<br />
mice, and identified a wide spectrum <strong>of</strong> cardiovascular malformations, which led to perinatal death <strong>of</strong> Ezh2<br />
mutant mice. In the Ezh2 mutant heart, the endocardial cushions (ECs) were hypoplastic and displayed<br />
impaired endothelial-to-mesenchymal transition (EMT). The mutant heart also exhibited decreased<br />
cardiomyocyte proliferation and increased apoptosis. We further identified that the Hey2 gene, which is<br />
important for cardiomyocyte proliferation and cardiac morphogenesis, is a downstream target <strong>of</strong> Ezh2. The<br />
regulation <strong>of</strong> Hey2 expression by Ezh2 may be independent <strong>of</strong> Notch signaling activity. Our work defines an<br />
indispensible role <strong>of</strong> the chromatin remodeling factor Ezh2 in normal cardiovascular development. This work<br />
was funded by grants HL101336-01(J.W.) from the National Institutes <strong>of</strong> Health, 09BGIA2050016 (J.W.) and<br />
11POST5680013 (L.C.) from the American Heart Association.<br />
2.15 QUANTIFYING TISSUE HYPOXIA, HYPOXIC STRESS AND BIOLOGICAL RESPONSES IN THE<br />
DEVELOPING EMBRYO<br />
Doreswamy Kenchegowda, Hongbin Liu, Steven A. Fisher<br />
Depts <strong>of</strong> Medicine (Cardiology) and Physiology, <strong>University</strong> <strong>of</strong> Maryland, Baltimore MD<br />
Reduced oxygen concentrations (tissue hypoxia) and oxygen gradients have been identified and proposed to<br />
play a role in heart morphogenesis. Further reductions in oxygen supply, as may occur with vascular<br />
insufficiency, high altitudes and anemia, have been linked to congenital heart defects. Critical deficiencies in<br />
this field <strong>of</strong> research include 1) quantifying where and under what conditions tissue hypoxia occurs and 2)<br />
linking transcriptional and morphogenic responses to endogenous and evoked hypoxia. For a quantitative<br />
approach to tissue hypoxia we have begun to study the ODD-Luc mouse in which Luciferase containing an<br />
Oxygen Degradation Domain (ODD) is expressed from the Rosa locus resulting in the accumulation <strong>of</strong><br />
Luciferase under hypoxic conditions. Luciferase activity is 10-fold higher in the E15.5 embryo as compared to<br />
adult tissues consistent with reduced oxygen concentrations. Exposure <strong>of</strong> E9.5 mice to hypoxic stress (8% O2<br />
for 4 hrs) increased luciferase activity by 2 fold in the embryos and much less in the mother’s tissues. We have<br />
previously observed that this level <strong>of</strong> hypoxic stress, in combination with the iron chelator DMOG, induces a<br />
hypoxic transcriptional response in the embryonic (St25) chick heart. Transcriptional pr<strong>of</strong>iling in the embryonic<br />
mouse heart is in progress. We conclude that ODDLuc provides a quantitative measure <strong>of</strong> tissue hypoxia in the<br />
developing heart. The mouse embryo develops under significant tissue hypoxia and is particularly susceptible to<br />
oxygen deprivation early in development.<br />
2.16 An essential role for RNA binding protein Bicc1 in cardiac development and function<br />
Hisato Yagi, Xiaoqin Liu, Bishwanath Chatterjee, You Li, Ashok Srinivasan, Andy Kim, Debby Farkas, Cecilia W.<br />
Lo<br />
Dept <strong>of</strong> Developmental Biology, <strong>University</strong> <strong>of</strong> Pittsburgh School <strong>of</strong> Medicine<br />
A mutant named Destro was recovered from our ongoing mouse ENU mutagenesis and found to have complex<br />
congenital cardiovascular defects (CHD) associated with heterotaxy - the randomized patterning <strong>of</strong> the left-right<br />
body axis. Some mutants exhibit complete mirror reversal <strong>of</strong> heart and visceral organ situs, but such mutants<br />
do not have CHD. Mutants also can have cysts associated with the kidney, biliary duct and pancreas. Using<br />
whole genome sequencing, we identified the mutation as Bicc1 c.606+2T>C , a splicing mutation causing in-frame<br />
deletion <strong>of</strong> 18 amino acids in the K homology domain involved in RNA recognition. This result is consistent with<br />
genome scanning analysis which mapped the mutation to the interval <strong>of</strong> mouse chromosome 10 containing<br />
Bicc1. Genotyping analysis showed some homozygous mutants with situs solitus are adult viable, but<br />
transthoracic echocardiography revealed cardiac function markedly declined with age. This was associated with<br />
dilated cardiomyopathy and marked increase in the expression <strong>of</strong> heart failure genes such as osteopontin.<br />
Interestingly, mapping analysis by genome scanning suggested linkage <strong>of</strong> a modifier locus on chromosome 5<br />
95
affecting term viability <strong>of</strong> mutants, an interval containing polycystin-2 and osteopontin. As Bicc1 is known to<br />
regulate polcystin-2 expression via modulation <strong>of</strong> mir-17 activity, this would suggest Bicc1 may be required for<br />
maintenance <strong>of</strong> cardiomyocyte homeostasis through microRNA regulation <strong>of</strong> cardiomyocyte<br />
differentiation/function. This may involve miR-539, as consensus binding site for this microRNA is found in both<br />
polycystin-2 and osteopontin. Overall, these findings suggest a novel role for Bicc1 in the modulation <strong>of</strong><br />
microRNAs involved in dilated cardiomyopathy. Supported by U01-HL098180.<br />
2.17 AKAP13 Rho-GEF and PKC-PKD Binding Domains Are Not Required for Development But Are for a<br />
Cardiac Functional Response to Chronic β-Adrenergic Stimulation<br />
Matthew J. Spindler 1 , Yu Huang 1 , Edward C. Hsiao 1,2 , Nathan Salomonis 1 , Mark J. Scott 1 , Graeme K.<br />
Carnegie 7 , Deepak Srivastava 1,3,4 , and Bruce R. Conklin 1,5,6<br />
1 Gladstone Institute <strong>of</strong> Cardiovascular Disease, San Francisco, CA; Departments <strong>of</strong> 2 Medicine, Division <strong>of</strong><br />
Endocrinology,<br />
3 Pediatrics,<br />
4 Biochemistry and Biophysics,<br />
96<br />
5 Medicine, and<br />
6 Cellular and Molecular<br />
Pharmacology, <strong>University</strong> <strong>of</strong> California, San Francisco, CA; 7 Department <strong>of</strong> Pharmacology, <strong>University</strong> <strong>of</strong> Illinois<br />
at <strong>Chicago</strong>, <strong>Chicago</strong>, IL<br />
Background: A-kinase anchoring proteins (AKAPs) are scaffolding molecules that coordinate and integrate Gprotein<br />
signaling events to regulate development, physiology, and disease. One family member, AKAP13,<br />
integrates Gs, Gq/11, and G12/13 signals through its Rho-guanine nucleotide exchange factor (Rho-GEF) domain<br />
and by binding protein kinase A, C (PKC), and D (PKD). AKAP13 appears to be required for development as<br />
null mouse embryos die by E10.5 with cardiovascular phenotypes. Additionally, the AKAP13 Rho-GEF and<br />
PKC-PKD binding domains mediate cardiomyocyte hypertrophy in cell culture. However, the requirements for<br />
the Rho-GEF and PKC-PKD binding domains during development and cardiac hypertrophy are unknown.<br />
Methodology/Principal Findings: To determine whether AKAP13 Rho-GEF activity and AKAP13-bound PKC-<br />
PKD are required for development, we generated mice that lacked the Rho-GEF and/or the PKC–PKD binding<br />
domains using gene-trap mutational events. Surprisingly, mutant mice were obtained at Mendelian ratios, had<br />
normal viability and were fertile. Adult mutant mice also had normal cardiac structure and electrocardiograms.<br />
To determine the role <strong>of</strong> these domains during chronic β-adrenergic-induced cardiac hypertrophy, we stressed<br />
mice with isoproterenol. We found that mice lacking the Rho-GEF and PKC-PKD binding domains had the same<br />
increase in heart size as control mice. However, these mutant hearts failed to increase cardiac ejection fraction<br />
or fractional shortening. Conclusions: Our results indicate that the AKAP13 Rho-GEF and PKC-PKD binding<br />
domains are not required for normal mouse development, cardiac architecture or function, or the structural<br />
response to β-adrenergic-induced cardiac hypertrophy. However, these domains are necessary for the cardiac<br />
functional response to chronic β-adrenergic stimulation.<br />
2.18 Optical coherence tomography captures rapid hemodynamic responses to acute hypoxia in the<br />
cardiovascular system <strong>of</strong> early embryos<br />
Shi Gu 1 , Michael W. Jenkins 1 , Lindsy M. Peterson 1 , Yong-Qiu Doughman 2 , Andrew M. Rollins 1 , Michiko<br />
Watanabe 2<br />
1 Department <strong>of</strong> Biomedical Engineering, Case Western Reserve <strong>University</strong>, Cleveland, Ohio 44106<br />
2 Department <strong>of</strong> Pediatrics, Case Western Reserve <strong>University</strong>, Cleveland, Ohio 44106<br />
Background: The trajectory to heart defects may start in tubular and looping heart stages when detailed analysis<br />
<strong>of</strong> form and function is difficult by currently available methods. We used a novel method, Doppler optical<br />
coherence tomography (OCT), to follow changes in cardiovascular function in quail embryos during acute<br />
hypoxic stress. Chronic fetal hypoxia is a known risk factor for congenital heart diseases (CHDs). Decreased<br />
fetal heart rates during maternal obstructive sleep apnea suggest that studying fetal heart responses under<br />
acute hypoxia is warranted. Results: We captured responses to hypoxia at the critical looping heart stages.<br />
Doppler OCT revealed detailed vitelline arterial pulsed Doppler waveforms. Embryos tolerated 1 hr <strong>of</strong> hypoxia<br />
(5%, 10%, or 15% O2), but exhibited changes including decreased systolic and increased diastolic duration in 5<br />
min. After 5 min, slower heart rates, arrhythmic events and an increase in retrograde blood flow were observed.<br />
These changes suggested slower filling <strong>of</strong> the heart, which was confirmed by four-dimensional Doppler imaging<br />
<strong>of</strong> the heart itself. Conclusions: Doppler OCT is well suited for rapid noninvasive screening for functional<br />
changes in avian embryos under near physiological conditions. Analysis <strong>of</strong> the accessible vitelline artery<br />
sensitively reflected changes in heart function and can be used for rapid screening. Acute hypoxia caused rapid<br />
hemodynamic changes in looping hearts and may be a concern for increased CHD risk. (Grant support:<br />
RO1HL083048, R01HL095717, T32EB007509, C06RR12463-01)
2.19 The Transcription Factor PPARδ may be involved in Cardiac Morphogenesis<br />
Yasuhiro Kosaka 1 , George Lezin 1 , Kevin J. Whitehead 2 , Katarzyna A. Cieslik 4 , Liliane Michalik 5 , Walter Wahli 5 ,<br />
H. Joseph Yost 3 , and Luca Brunelli 1 .<br />
1 Department <strong>of</strong> Pediatrics (Neonatology), 2 Internal Medicine (Cardiology), and 3 Neurobiology and Anatomy,<br />
<strong>University</strong> <strong>of</strong> Utah, Salt Lake City, UT; 4 Department <strong>of</strong> Medicine, Baylor College <strong>of</strong> Medicine, Houston, TX;<br />
5 Center for Integrative Genomics, <strong>University</strong> <strong>of</strong> Lausanne, Lausanne, Switzerland<br />
Deletion <strong>of</strong> Ppard, encoding the ligand-inducible nuclear receptor peroxisome proliferator-activated receptor δ<br />
(PPARδ), in mice is believed to cause mid-gestation death as a result <strong>of</strong> placental defects, but cardio-specific<br />
deletion <strong>of</strong> the gene with α-MyHC-Cre leads to post-natal cardiomyopathy and death by 4-10 months <strong>of</strong> age. In<br />
addition, PPARδ is ubiquitously expressed, including the embryonic heart, and we have shown that it modulates<br />
transcription <strong>of</strong> the 14-3-3ε protein (Ywhae), which in turn regulates ventricular morphogenesis. Thus, we<br />
hypothesized that PPARδ might be involved in heart development. We confirmed that PPARδ is expressed in<br />
the heart during development, and showed that the PPARδ-specific agonist L-165,041 upregulates 14-3-3ε<br />
levels in P19CL6 cells differentiating toward the cardiomyocyte lineage and primary cardiomyocytes.<br />
Administration <strong>of</strong> 30 mg/kg/day L-165,041 for three days to pregnant females si gnificantly upregulated Ywhae<br />
mRNA levels in E12.5 wildtype and Ywhae+/- embryonic hearts. We also found that Ppard-/- mice present<br />
significant ventricular dilatation at E9.5, and we are currently deleting Ppard with a cTNT-Cre driver and<br />
performing ECHOs in E9.5 Ppard-/- mice to evaluate both umbilical artery flow and cardiac function to better<br />
assess the relative role <strong>of</strong> PPARδ in the placenta and heart. These results indicate that PPARδ modulates<br />
cardiac 14-3-3ε, a regulator <strong>of</strong> ventricular morphogenesis, and suggest that PPARδ may directly control heart<br />
development in mice.<br />
2.20 Mechanisms that control the function <strong>of</strong> the mitochondrial permeability transition pore during<br />
cardiac development and myocyte differentiation<br />
George A. Porter Jr., MD, PhD 1,2,3 , Jennifer R. Hom, PhD 1<br />
1 Department <strong>of</strong> Pediatrics, 2 Department <strong>of</strong> Pharmacology and Physiology, and 3 Aab Cardiovascular Research<br />
Institute, <strong>University</strong> <strong>of</strong> Rochester School <strong>of</strong> Medicine, Rochester, NY.<br />
We recently found that changes in mitochondria in the embryonic mouse heart are regulated by the<br />
mitochondrial permeability transition pore (mPTP) and closure <strong>of</strong> the mPTP decreased oxidative stress that<br />
facilitates cardiac myocyte differentiation. These experiments suggested that the changes in the expression<br />
and/or function <strong>of</strong> mPTP-regulatory protein, CyP-D, regulate mPTP function and myocyte differentiation in the<br />
early heart. To test this hypothesis, early embryonic hearts were harvested from wild-type (WT) and CyP-D null<br />
mice for analysis <strong>of</strong> whole hearts and primary myocyte cultures. Mitochondrial and mPTP function and myocyte<br />
differentiation were examined using biochemical assays and fluorescence microscopy. Gene and protein<br />
expression and post-translational modification was assessed using RNASeq, immunoblotting (IB),<br />
immun<strong>of</strong>luorescence microscopy (IF) and infection <strong>of</strong> myocytes with WT and mutated CyP-D expression<br />
vectors. Between E9.5 and 11.5, expression <strong>of</strong> CyP-D and other components <strong>of</strong> the mPTP generally increased<br />
by 20%. IB experiments showed similar small changes in protein expression, while IF <strong>of</strong> sectioned hearts<br />
demonstrated increased CyP-D expression in the less differentiated cells <strong>of</strong> the ventricular walls compared to<br />
the more differentiated trabeculae. Re-expression <strong>of</strong> WT CyP-D in CyP-D null myocytes blocked the maturation<br />
<strong>of</strong> mitochondrial structure and function and the increased myocyte differentiation we have previously observed<br />
in CyP-D null hearts. In contrast, expression <strong>of</strong> inactive CyP-D had no effect, suggesting that the peptidyl-prolyl<br />
isomerase activity <strong>of</strong> CyP-D is important for these processes. These studies suggest the activity <strong>of</strong> CyP-D<br />
regulates the mPTP and plays a critical role in cardiac myocyte differentiation in the embryonic heart.<br />
2.21 A SENP5-Dependent deSUMOylation Pathway for Dilated Cardiomyopathy<br />
Eun Young Kim 1,2 , Wei Yu 3 , Robert J. Schwartz 1,3 , Jun Wang 1<br />
1 Center for Stem Cell Engineering, Texas Heart Institute, 2 Gene and Development Program,<br />
<strong>University</strong> <strong>of</strong> Texas Health Science Center at Houston, Houston TX 77030, USA,<br />
3 Department <strong>of</strong> Biochemistry, <strong>University</strong> <strong>of</strong> Houston, Houston, TX 77204, USA<br />
SUMOylation <strong>of</strong> proteins regulates diverse cellular processes related to transcription, apoptosis, cell cycle, and<br />
protein stability. Sentrin protease 5 (SENP5) is a SUMO2/3 specific deconjugation enzyme and its functional<br />
roles in the cardiac pathophysiology remain unknown. Here we show that transgenic mice expressing SENP5 in<br />
the heart (SENP5 Tg) develop dilated cardiomyopathy and heart failure that mimic human heart disease with<br />
fibrosis, and impairment in myocardial function. Elevated apoptosis and reduced cell proliferation were also<br />
97
observed in the hearts <strong>of</strong> these animals. Surprisingly, the hearts from SENP5 Tg mice have markedly reduced<br />
mitochondria enzymatic activities due to almost complete loss <strong>of</strong> mitochondrial cristae, and compromised<br />
cardiac energy metabolism. Moreover, the Ca2+ deposition was significantly enhanced in SENP5 Tg mouse<br />
hearts. These findings identify that a novel SENP5-mediated deSUMOylation pathway controls mitochondrial<br />
function and concomitant pat hological remodeling <strong>of</strong> the heart. Finally we also observed increased level <strong>of</strong><br />
SENP5 in human failing hearts. Thus a pharmacologic intervention <strong>of</strong> SENP5 might be a promising therapeutic<br />
approach to prevent dilated cadiomyopathy and heart failure.<br />
2.22 The role <strong>of</strong> PDGF signaling on zebrafish heart regeneration<br />
Jieun Kim, Ying Huang and Ching-Ling Lien<br />
The Saban Research Institute, Children’s Hospital Los Angeles, CA, USA<br />
In contrast to mammalian adult hearts, zebrafish hearts regenerate their damaged hearts. To date, cardiac injury<br />
methods utilized to study zebrafish heart regeneration include 20% ventricular resection, cryoinjury and genetic<br />
ablation. It remains to be determined if zebrafish hearts respond differently to different types <strong>of</strong> injury. A major<br />
difference between cryoinjury and amputation is that dead cells and tissues remain in the heart after cryoinjury,<br />
mimicking the pathogenesis after myocardial infarction. It was reported that scar tissue does form after<br />
cryoinjury, but it later absorbed and zebrafish hearts still regenerate within 60-120 days post cryoinjury (dpc).<br />
However, we observed that 30-50% <strong>of</strong> zebrafish hearts still have collagen scar by 120dpc. The discrepancy<br />
between our data ad the published report might result from initial injury sizes and suggests that a threshold for<br />
cryo damage might exist for the regenerative capacity <strong>of</strong> zebrafish heart. We previously showed that blocking<br />
PDGFR signaling decreases epicardial cell proliferation, epithelial-to-mesenchymal transition, and new coronary<br />
vessel formation during regeneration after amputation. Using transgenic fish expressing a dominant-negative<br />
form <strong>of</strong> PDGFRβ, we will determine the functions <strong>of</strong> PDGF signaling in heart regeneration after cryoinjury.<br />
2.23 Role <strong>of</strong> LEK1 in cardiac disease and function<br />
Paul M. Miller, Ellen Dees, David Bader<br />
Vanderbilt <strong>University</strong> Medical Center<br />
In our lab, we have developed global as well as heart-specific CENPF-/- (LEK1) mouse models. Utilizing our<br />
heart-specific model, we have found that LEK1 mutation leads to a myriad <strong>of</strong> problems including: an overall<br />
decrease in intercalated disc number, significant fibrosis, erratic ECG patterns, progressive dilated<br />
cardiomyopathy, and a 20% chance <strong>of</strong> sudden death. To date, the molecular mechanism underlying LEK1<br />
involvement in these processes remains unknown. Although LEK1 is a very large and multi-functional protein,<br />
one common functional theme has emerged in the literature: the interaction <strong>of</strong> LEK1 with the microtubule<br />
cytoskeleton. Previous studies have identified an important role for LEK1 in microtubule nucleation at the<br />
centrosome as well regulation <strong>of</strong> vesicular transport and cell coupling. Here, using CENPF-/- mouse embryonic<br />
fibroblasts, we show that directionally persistent migration as well as overall microtubule network organization is<br />
disrupted. Our overall hypothesis is that cytoskeletal defects mediated by LEK1 mutation result in<br />
developmental, morphogenic, and functional abnormalities in the heart.<br />
Section 3: Genomics and Gene Regulation<br />
S3.1 Isolation <strong>of</strong> nuclei from specific cardiac lineages in zebrafish for genome-wide and epigenomic<br />
analyses<br />
Bushra Gorsi and H. Joseph Yost<br />
Department <strong>of</strong> Neurobiology and Anatomy, <strong>University</strong> <strong>of</strong> Utah Molecular Medicine Program, Eccles Institute <strong>of</strong><br />
Human Genetics, Building 533, Room 3160, 15 North 2030 East, Salt Lake City, Utah 84112-5330, USA.<br />
To understand the critical gene regulatory interactions that drive atrial versus ventricle identity, we are using<br />
genome-wide analysis <strong>of</strong> differentiating cardiomyocytes to uncover changes in epigenetic and transcriptome<br />
pr<strong>of</strong>iles. Although advances in sequencing technology have greatly enhanced our ability to characterize<br />
genome-wide changes in epigenetic modifications and gene expression, this has been constrained by the<br />
technical challenges faced in purifying specific cell lineages in adequate amounts from developing systems.<br />
Therefore we are developing a novel approach that will allow us to isolate nuclei <strong>of</strong> differentiating<br />
cardiomyocytes in-vivo. We have created transgenic lines in which a biotin ligase recognition peptide (BLRP) is<br />
fused to an outer membrane nuclear envelope protein (NEP). Regulated expression <strong>of</strong> BirA enzyme drives the<br />
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in vivo biotinylation <strong>of</strong> BLRP-NEP, allowing purification <strong>of</strong> nuclei with streptavidin-conjugated beads. When BirA<br />
is ubiquitously expressed, we can successfully elute chromatin threefold higher from transgenic embryos with<br />
biotinylated nuclei than transgenic embryos devoid <strong>of</strong> BirA enzyme. We successfully performed ChIP with<br />
multiple histone marks, indicating that the chromatin within these purified nuclei is useful for epigenomic<br />
analyses. We are creating double transgenics in which BLRP-NEP is biotinylated in specific heart lineages in<br />
order to capture chromatin and nuclear RNA (mRNA and miRNA) from cardiac progenitors, ventricle precursors<br />
and atrial precursors. This will allow us to perform genome-wide analysis <strong>of</strong> epigenetic and transciptome pr<strong>of</strong>iles<br />
in specific subsets <strong>of</strong> lineages and tissues during any stage <strong>of</strong> zebrafish cardiac development. Supported in<br />
part by U01HL098179 (NHLBI Bench-to-Bassinet program)<br />
S3.2 TU-tagging in mice defines dynamic gene expression programs in specific cardiovascular or other<br />
cell types within intact tissues<br />
Leslie Gay 1-3 , Michael R. Miller 1-3 , Vidusha Devasthali 2 , P. Brit Ventura 2 , Zer Vue 4 , Heather L. Thompson 4 , Sally<br />
Temple 5 , Hui Zong 2 , Michael D. Cleary 4 , Kryn Stankunas 2 , Chris Q. Doe 1-3<br />
Institute <strong>of</strong> Neuroscience 1 , Institute <strong>of</strong> Molecular Biology 2 , Howard Hughes Medical Institute 3 , <strong>University</strong> <strong>of</strong><br />
Oregon, Eugene, OR 97403. 4 Division <strong>of</strong> Natural Sciences, <strong>University</strong> <strong>of</strong> California, Merced, CA 95340.<br />
5 Neural Stem Cell Institute, Rensselaer, NY 12144<br />
Changes in gene expression programs underlie all aspects <strong>of</strong> cardiovascular development and disease.<br />
Therefore, transcriptional pr<strong>of</strong>iling is a powerful and direct approach to study these and other biological<br />
processes. Current methods are limited by difficulties isolating specific cell populations from complex tissues,<br />
changes in gene expression that occur during sample preparation, and the inability to distinguish dynamic<br />
transcriptional changes within a cellular pool <strong>of</strong> pre-existing RNA. To overcome these limitations in mouse<br />
research, we have developed a method called “TU-tagging” that allows for purification <strong>of</strong> newly transcribed RNA<br />
from defined cell types. With TU-tagging, genetic and chemical methods intersect to provide spatiotemporal<br />
controlled RNA labeling in vivo. Cre-induced expression <strong>of</strong> UPRT using a newly-developed transgenic mouse<br />
(CA:loxStoploxUPRT) directs the incorporation <strong>of</strong> injected 4-thiouracil (4TU) into nascent RNA, generating a<br />
pulse <strong>of</strong> cell type-specific thiolated RNA. The thio-RNA is readily purified from whole tissue RNA preparations<br />
and RNA-seq is used to define “acute” transcriptomes. We validate mouse TU-tagging by using<br />
Tie2:Cre;CA:loxStoploxUPRT double transgenic mice to purify endothelial RNA from brain or<br />
endothelial/endocardial RNA from heart at both embryonic and postnatal stages. In each case, the vast majority<br />
<strong>of</strong> the most enriched transcripts are verified as being expressed in Tie2:Cre marked cell lineages. We define a<br />
core set <strong>of</strong> pan-endothelial transcripts and identify groups <strong>of</strong> endothelial/endocardial lineage transcripts with<br />
shared specific expression patterns. TU-tagging provides a novel, sensitive method for characterizing<br />
transcriptome dynamics in rare cell types within the intact mouse.<br />
S3.3 Escape <strong>of</strong> Gene Body DNA Methylation in Cardiac Genes is Cooperative with the Cell Type-specific<br />
Expression Patterns<br />
Mayumi Oda, Shinji Makino, Hirokazu Enomoto, Ruri Kaneda, Shinsuke Yuasa and Keiichi Fukuda.<br />
Center for Integrated Medical Research, School <strong>of</strong> Medicine, Keio <strong>University</strong>,Tokyo 160-8582 JAPAN.<br />
Mammalian genome is highly methylated in C-G dinucleotides (CpGs), over 60% <strong>of</strong> which are believed to be<br />
methylated. The effect <strong>of</strong> DNA methylation has been investigated intensively in the promoter regions, but it has<br />
still been unknown in non-promoter regions, especially in the cell type-specific effect. Although we previously<br />
reported that gene body DNA methylation levels are correlated with the transcription levels and the replication<br />
timing in human cell lines (Suzuki et al. Genome Res 2011), function <strong>of</strong> gene body DNA methylation levels in<br />
developing cells is unknown. In this study, we focused on the DNA methylation pattern in cardiomyocytes as the<br />
established differentiated cells. We examined genome-wide DNA methylation pr<strong>of</strong>iles <strong>of</strong> cardiomyocytes and<br />
non-cardiomyocyte cells from different developmental stages by the deep sequencing technique, and found that<br />
the gene body regions <strong>of</strong> some cardiac genes, including myosin heavy chain (MHC) genes, are less methylated<br />
than other genes, where the DNA methylation levels were dramatically decreased in the latter half <strong>of</strong> pregnancy.<br />
To evaluate the effect <strong>of</strong> gene body DNA methylation, we performed luciferase reporter assay by the<br />
reconstruction using the SssI-methylated and unmethylated gene body fragments, and confirmed that the gene<br />
body DNA methylation is repressive in the gene expression, mostly at the transcriptional level. Collectively, the<br />
gene body DNA methylation levels in some cardiac genes were developmentally regulated and possibly<br />
enhance the transcription <strong>of</strong> cardiac genes.<br />
99
S3.4 The novel mechanism <strong>of</strong> histone-chromatin regulation for cardiomyocytes regeneration<br />
Ryo Nakamaura, 1 Kazuko Koshiba-Takeuchi, 1 Yuko Tsukahara, 1 Tetsuo Sasano, 3 Mizuyo Kojima, 1 Tetsushi<br />
Furukawa, 3 Hesham A Sadek, 4 Jun K Takeuchi, 1,2<br />
1 Univ <strong>of</strong> Tokyo, 2 JST PRESTO, 3 Tokyo Medical and Dental Univ, 4 Univ <strong>of</strong> Texas Southwestern Medical Center<br />
Fishes and amphibians have high capacities for heart regeneration even in the adult, whereas we mammalians<br />
lose such regeneration capacities. Understanding the mechanism <strong>of</strong> heart regeneration should provide the most<br />
important cues on the therapies for human heart failure. We found that SWI/SNF-BAF type cardiac chromatin<br />
remodeling factors and histone regulators were strongly up-regulated within 12 hours after resection <strong>of</strong> ventricle<br />
both in neonatal mice and axolotl, and these expression was maintained for one week during regeneration. To<br />
address whether these factors function as the early response factors in mammalian heart regeneration, we<br />
constructed BAF-overexpression (BAF-TG) mice, which showed stable expression <strong>of</strong> BAFs even in the adult<br />
heart. Interestingly, BAF-TG prevented fibrosis post myocardial infarction and regenerated their injured parts<br />
with the proliferation <strong>of</strong> cardiomyocytes. In vivo ChIP analyses revealed that the SWI/SNF core factor, Brg1,<br />
directly regulated several cardiac fetal genes’ promoters such as Nppa, Tnnt2, Myl7 only at the embryonic<br />
stages. Surprisingly, in the BAF-TG, major cardiac fetal genes’ promoters were still opened in the adult state.<br />
And si-treatment <strong>of</strong> polycomb or repress-type chromatin factor s increased cell proliferation in vitro. These data<br />
demonstrate that histone-chromatin conformation is changed depending on the developmental stages on<br />
several cardiac genes relating with the regeneration capacity.<br />
S3.5 Epigenetic regionalization <strong>of</strong> the developing mouse heart<br />
Scott Smemo*, Noboru J. Sakabe*, Ivy Aneas, Marcelo A. Nobrega<br />
Department <strong>of</strong> Human Genetics, <strong>University</strong> <strong>of</strong> <strong>Chicago</strong>, <strong>Chicago</strong>, IL.<br />
A multitude <strong>of</strong> differences in gene expression have been described and correlated with cardiac chamber identity,<br />
structure, function and disease. Nonetheless, it is not fully resolved how these transcriptional spatial<br />
asymmetries, evident at even the very early stages <strong>of</strong> morphogenesis, are established and maintained. An<br />
intriguing hypothesis is that these asymmetries are contributed to by differences in chromatin accessibility,<br />
which potentiate or exclude expression <strong>of</strong> specific genes. By performing transcriptional pr<strong>of</strong>iling by RNA-seq on<br />
isolated left and right ventricles <strong>of</strong> embryonic mice and correlating the findings with ventricular ChIP-seq data for<br />
H3K27me3, a mark <strong>of</strong> closed chromatin associated with gene repression, and H3K4me1, a histone modification<br />
associated with open chromatin and active genes, we observe a significant subset <strong>of</strong> chamber-specific cardiac<br />
genes, including Tbx5, that are selectively marked by both H3K4me1 and H3K27me3 in opposite chambers. In<br />
the left ventricle, these genes are functionally linked to roles in energy metabolism, whereas in the right<br />
ventricle, coordinately marked genes are associated with developmental roles. We demonstrate that Tbx5<br />
enhancers have the potential <strong>of</strong> driving expression in broad cardiac domains, and that the restricted Tbx5<br />
expression is mainly due to regionalization <strong>of</strong> epigenetic marks. Furthermore, this spatially regionalized pattern<br />
<strong>of</strong> gene repression is redeployed to limit Tbx5 expression to forelimb and not hindlimb, highlighting a general<br />
chromatin-based mechanism to affect spatially-restricted expression. Beyond our general understanding <strong>of</strong> how<br />
the developing heart is patterned, these findings have implications for efforts at directed differentiation <strong>of</strong> stem<br />
cells into cardiomyocytes for therapeutic uses.<br />
S3.6 Drosophila microRNA-1 Genetically Interacts with little imaginal discs, a histone demethylase.<br />
Isabelle N. King 1,2<br />
1 Gladstone Institute <strong>of</strong> Cardiovascular Disease, 1650 Owens Street, San Francisco, CA 94158, 2 Department <strong>of</strong><br />
Pediatrics, <strong>University</strong> <strong>of</strong> California, San Francisco, San Francisco, CA, 94143.<br />
The expression pattern and function for the microRNA miR-1 is conserved in both invertebrates and vertebrates,<br />
suggesting that it modulates some <strong>of</strong> the most fundamental processes within cardiac and skeletal muscle,<br />
including Notch signaling. We performed a forward genetic screen in Drosophila that identified little imaginal<br />
discs (lid), a chromatin-remodeling enzyme, as a genetic partner <strong>of</strong> dmiR-1. Lid demethylates histone H3 lysine<br />
4 (H3K4), thereby epigenetically decreasing the activity <strong>of</strong> selected promotors. Lid forms a complex with Notch<br />
pathway regulatory proteins, and loss <strong>of</strong> lid activity results in de-repression <strong>of</strong> Notch downstream targets. We<br />
have found that in flies, heterozygosity <strong>of</strong> lid in the context <strong>of</strong> dmiR-1 overexpression resulted in exacerbation <strong>of</strong><br />
the dmiR-1 overexpression phenotype, consistent with de-repression <strong>of</strong> Notch signaling. Furthermore, we<br />
discovered that use <strong>of</strong> a balancer chromosome containing a mutation in the Notch ligand Serrate, unmasked an<br />
indirect flight muscle phenotype in lid (-/-) flies that was modified upon manipulation <strong>of</strong> dmiR-1 levels. These<br />
100
esults imply that dmiR-1 may selectively modulate the epigenetic modification <strong>of</strong> histones at Notch-responsive<br />
loci during fly heart and muscle development.<br />
3.7 Mutual Regulation <strong>of</strong> Nkx2.5 and Fibulin-1 in the SHF Regulatory Network<br />
Anthony J. Horton 1 , Marion A. Cooley 2 , Waleed O. Twall 2 , Victor M. Fresco 2 , Boding Zhang 1 , Christopher D.<br />
Clark 1 , W. Scott Argraves 2 and Kyu-Ho Lee 1*<br />
1 Children’s Hospital, Division <strong>of</strong> Pediatric Cardiology and 2 Department <strong>of</strong> Regenerative Medicine and Cell<br />
Biology, Medical <strong>University</strong> <strong>of</strong> South Carolina, Charleston, SC 29425<br />
Approximately 12,000 children are born yearly with malformations <strong>of</strong> the aorta and/or pulmonary artery resulting<br />
from abnormal outflow tract (OFT) development. The cardiac OFT arise from progenitor cells <strong>of</strong> the second<br />
heart field (SHF) and animal studies have shown that this process is subject to the influence <strong>of</strong> several<br />
transcription factor, growth factor and extracellular matrix (ECM) genes. It is therefore likely that the<br />
combinatorial effects <strong>of</strong> multiple gene mutations underlie genetically complex OFT congenital heart defects<br />
(CHD) entities in humans. As part <strong>of</strong> an effort to determine functional linkages between known OFT<br />
developmental regulators, we have found evidence for mutual regulation <strong>of</strong> the cardiac transcription factor<br />
Nkx2.5 and the ECM protein fibulin-1 (Fbln1), both <strong>of</strong> which are required for normal SHF and OFT development<br />
in mice. Specifically we have found that Fbln1 expression levels are decreased in SHF regions in Nkx2.5 null<br />
mice, and that Nkx2.5 appears to regulate Fbln1 expression in the SHF via binding to an evolutionarily<br />
conserved NKE binding site in promoter proximal regions. Conversely, Fbln1 expression reciprocally regulates<br />
Nkx2.5, as Nkx2.5 expression is decreased in SHF regions <strong>of</strong> Fbln1 null mice in association with increased<br />
expression <strong>of</strong> a potential direct repressor <strong>of</strong> Nkx2.5, the growth factor activated transcription factor Egr1. These<br />
findings support the hypothesis that Nkx2.5 and Fbln1 are key components <strong>of</strong> a growth factor-mediated<br />
homeostatic mechanism regulating SHF proliferation and OFT development.<br />
3.8 FOG-2 Mediated Recruitment <strong>of</strong> the NuRD Complex Regulates Cardiomyocyte Proliferation during<br />
Heart Development<br />
Audrey Jerde, Zhiguang Gao, Spencer Martens, Judy U. Early, Eric C. Svensson<br />
Department <strong>of</strong> Medicine, The <strong>University</strong> <strong>of</strong> <strong>Chicago</strong>, <strong>Chicago</strong>, IL, 60637<br />
FOG-2 is a multi-zinc finger protein that binds GATA4 and modulates GATA4-mediated transcriptional control <strong>of</strong><br />
target genes during heart development. Our previous work has demonstrated that the Nucleosome Remodeling<br />
and Deacetylase (NuRD) complex physically interacts with FOG-2 and is necessary for FOG-2 mediated<br />
repression <strong>of</strong> GATA4 activity in vitro. In this report, we describe the generation and characterization <strong>of</strong> mice<br />
homozygous for a mutation that disrupts FOG-2/NuRD complex interaction (FOG-2 R3K5A ). These mice exhibit a<br />
perinatal lethality and have multiple cardiac malformations, including a ventricular septal defect and a thin<br />
ventricular myocardium. To investigate the mechanism underlying the thin ventricular walls, we measured the<br />
rate <strong>of</strong> cardiomyocyte apoptosis and proliferation in wild-type and FOG-2 R3K5A developing hearts. We found no<br />
significant difference in the rate <strong>of</strong> cardiomyocyte apoptosis between wild-type and FOG-2 R3K5A mice. However,<br />
cardiomyocyte proliferation was reduced by 29.8% in FOG-2 R3K5A mice. Interestingly, we found by gene<br />
expression analysis that the cell cycle inhibitor p21 is up-regulated 1.7-fold in FOG-2 R3K5A hearts. Further, we<br />
demonstrate that FOG-2 can directly repress the activity <strong>of</strong> the p21 gene promoter using an in vitro transient<br />
transfection assay. In addition, chromatin immunoprecipitation (ChIP) reveals that the NuRD complex is bound<br />
to the p21 promoter in wild-type embryonic day 14.5 hearts but is diminished at this promoter in mutant hearts.<br />
Taken together, our results suggest the FOG-2/NuRD interaction is required for cardiomyocyte proliferation by<br />
specifically down-regulating expression <strong>of</strong> the cell cycle inhibitor p21 during heart development.<br />
3.9 A strict lineage boundary between the first and second heart fields is defined by the contribution <strong>of</strong><br />
the Tbx5 lineage.<br />
Joshua D. Wythe, W. Patrick Devine, Kazuko Koshiba-Takeuchi, Kyonori Togi, Benoit G. Bruneau<br />
Gladstone Institute <strong>of</strong> Cardiovascular Disease, <strong>University</strong> <strong>of</strong> California, San Francisco. 1650 Owens Street, San<br />
Francisco, CA 94158, USA.<br />
The theory that the heart arises from two distinct heart fields suggests important clues as to how the heart is<br />
patterned. Determining if contributions <strong>of</strong> distinct first heart field (FHF) and second heart field (SHF) cell<br />
populations to the fully formed heart exist is central to understanding heart development and the etiology <strong>of</strong><br />
congenital heart defects (CHDs). Haploinsufficiency <strong>of</strong> the transcription factor TBX5 causes Holt-Oram<br />
Syndrome, which includes CHDs. Tbx5 expression outlines a region <strong>of</strong> the heart that is presumed to correspond<br />
101
to the FHF, and Tbx5 is required in the posterior half <strong>of</strong> the heart. However, it is not known what structures<br />
within the heart the Tbx5 lineage gives rise to, or how these relate to the importance <strong>of</strong> Tbx5 in CHDs. Using an<br />
inducible Cre recombinase inserted at the Tbx5 locus, and various SHF genetic markers, we have mapped the<br />
Tbx5-expressing lineage and its intersection with the SHF, to discern field allocation in cardiac morphogenesis.<br />
We fi nd that from the earliest stages <strong>of</strong> Tbx5 expression, prior to formation <strong>of</strong> the linear heart tube, the Tbx5<br />
lineage contributes to the posterior segments <strong>of</strong> the heart, ending at a sharp boundary at the interventricular<br />
septum, with little overlap with the SHF. We propose that the earliest Tbx5 expressing cells define the FHF<br />
lineage, and that a strict lineage boundary exists between the FHF and SHF prior to morphogenic distinctions<br />
between their descendents. Understanding the contribution <strong>of</strong> Tbx5 to the FHF will contribute to our knowledge<br />
<strong>of</strong> heart development and CHDs.<br />
3.10 Tbx5-Hedgehog pathway is required in second heart field cardiac progenitors for atrial septation<br />
Linglin Xie 1, 2 , Andrew D. H<strong>of</strong>fmann 1 , Ozanna Burnicka-Turek 1 , Joshua M. Friedland-Little 1 , Ke Zhang 3 , Ivan P.<br />
Moskowitz 1<br />
1 Departments <strong>of</strong> Pediatrics and Pathology, The <strong>University</strong> <strong>of</strong> <strong>Chicago</strong>, <strong>Chicago</strong>, IL, 60637, USA; 2 Department <strong>of</strong><br />
Biochemistry and Molecular Biology and 3 Department <strong>of</strong> Pathology, The <strong>University</strong> <strong>of</strong> North Dakota, Grand<br />
Forks, ND, 58202, USA<br />
The developmental mechanisms underlying human Congenital Heart Disease (CHD) are poorly established.<br />
Atrial Septal Defects (ASDs), a common form <strong>of</strong> CHD, can result from haploinsufficiency <strong>of</strong> cardiogenic<br />
transcription factors including Tbx5. We demonstrate that Tbx5 is required outside the heart in second heart<br />
field (SHF) cardiac progenitors for atrial septation in mice. Conditional Tbx5 haploinsufficiency in the SHF, but<br />
not in the myocardium or endocardium, caused ASDs. Tbx5 SHF knockout embryos lacked atrial septum<br />
progenitors. We identified SHF Tbx5- responsive cell cycle progression genes, including cdk6 as a direct target,<br />
and Tbx5 mutant SHF progenitors demonstrated a mitotic defect. Genetic and molecular evidence including the<br />
rescue <strong>of</strong> atrial septation in Tbx5 mutant embryos by constitutive Hh-signaling placed Tbx5 upstream or parallel<br />
to Hh signaling in cardiac progenitors. We identified Gas1, a Hh-pathway member upstream <strong>of</strong> Smo, as a direct<br />
target <strong>of</strong> Tbx5. We also found that Osr1, required for atrial septaion in mice, is expressed in a Tbx5-dependent<br />
manner. We further identified Osr1 as a direct target <strong>of</strong> Tbx5 required for atrial septation. These results<br />
describe a SHF Tbx5-Hh-signaling network and independent Tbx5-Osr1 pathway required for atrial septation.<br />
Ongoing work is aimed at identifying molecular links between these pathways in atrial septation. A paradigm<br />
defining molecular requirements downstream <strong>of</strong> Tbx5 in cardiac progenitors as organizers <strong>of</strong> cardiac septum<br />
morphogenesis has implications for the ontogeny <strong>of</strong> CHD.<br />
3.11 The Six/Eya transcription complex in cardiac outflow tract development and disease<br />
JingYing Wang and Sean Xue Li<br />
Children's Hospital Boston, Harvard Medical School<br />
Background: The Six-family genes (Six1-6) encode the homeodomain-containing transcription factors. They<br />
physically interact with the Eya-family c<strong>of</strong>actors (Eya1-4) to form the canonical Six-Eya transcription complex.<br />
We recently reported that Six1 and Eya1 are critical for cardiac outflow tract development since compound<br />
mouse mutants exhibit severe outflow tract septation and alignment defects. It is unknown, however, whether<br />
any other members <strong>of</strong> the Six- and Eya-family genes are also involved in cardiovascular development. Results:<br />
Six2 is closely related to Six1 with over 95% sequence homology. To examine whether Six2 is involved in the<br />
cardiac outflow tract development, we examine its expression pattern, genetic lineages, and its functions in<br />
mouse mutants. Our findings demonstrate that Six2 is expressed in a subset <strong>of</strong> the second heart field<br />
progenitors, which primarily contribute to the pulmonary outflow trunk and the right ventricle. Compound<br />
Six2;Six1 and Six2;Eya1 mutants have a spectrum <strong>of</strong> cardiac outflow tract defects. Six2 expression is<br />
significantly down regulated in the Shh mutant, which exhibits severe pulmonary trunk agenesis phenotype. We<br />
find that Six2 + lineages are significantly diminished in Shh mutants, suggesting that Shh signaling regulates the<br />
behavior <strong>of</strong> the Six2 + progenitors during pulmonary trunk and right ventricle formation. Conclusions: Six2<br />
marks a critical subpopulation <strong>of</strong> the second heart field progenitors that give rise to primarily pulmonary but not<br />
aortic outflow trunk, and the Shh-Six2 genetic pathway is essential for pulmonary trunk and right ventricle<br />
formation. In addition, we will also present our preliminary targeted genomic screening <strong>of</strong> DiGeorge patients as<br />
we have identified putative mutations in the Six- and Eya-family genes. These new finding suggests, for the first<br />
time, that Six- and Eya-family genes are directly linked pathogenesis <strong>of</strong> human congenital heart defects.<br />
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3.12 PRC2 Regulates Heart Development and Adult Heart Energy Homeostasis.<br />
Aibin He 1 , Qing Ma 1 ,Jingjing Cao 1 , Alexander von Gise 1 , Pingzhu Zhou 1 , William T. Pu 1,2,<br />
1 Department <strong>of</strong> Cardiology, Children’s Hospital Boston and Harvard Medical School, Boston, MA<br />
2 Harvard Stem Cell Institute, Harvard <strong>University</strong>, Cambridge, MA<br />
Epigenetic marks are crucial for organogenesis, but their role in heart development and function is poorly<br />
understood. Polycomb Repressive Complex 2 (PRC2) trimethylates histone H3 at lysine 27, establishing<br />
H3K27me3 repressive epigenetic marks that promote tissue-specific differentiation by silencing ectopic gene<br />
programs. Firstly we studied the function <strong>of</strong> PRC2 in murine heart development using a tissue-restricted<br />
conditional inactivation strategy. Inactivation <strong>of</strong> the PRC2 subunit Ezh2 by Nkx2-5 Cre (Ezh2 NK ) caused lethal<br />
congenital heart malformations, namely compact myocardial hypoplasia, hypertrabeculation, and ventricular<br />
septal defect. Candidate and genome-wide RNA expression pr<strong>of</strong>iling and chromatin immunoprecipitation<br />
analyses <strong>of</strong> Ezh2 NK heart identified genes directly repressed by EZH2. Among these were the potent cell cycle<br />
inhibitors Ink4a/b, whose upregulation was associated with decreased cardiomyocyte proliferation in Ezh2 NK .<br />
EZH2-repressed genes were enriched for transcriptional regulators <strong>of</strong> non-cardiomyocyte expression programs,<br />
such as Pax6, Isl1, Six1. EZH2 was also required for proper spatiotemporal regulation <strong>of</strong> cardiac gene<br />
expression, such as Hcn4, Mlc2a, and Bmp10. Moreover, the cardiomyocytes restriced ablation <strong>of</strong> another<br />
PRC2 key component Eed at the neonatal stage caused dilated cardiomyopathy. Further study showed the<br />
mutant cardiomyocytes were defect in energy metabolism by increases in both glycolysis and mitochondria<br />
respiration, however without change in total ATP production. Glucokinase (GcK), a kinase for phosphorylation <strong>of</strong><br />
Glucose to phosphate-6-glucose in the first step <strong>of</strong> glycolysis, was largely upregulated in Eed mutant, at least<br />
partially contributing to the enhanced glycolysis. Taken together, our study reveals a previously undescribed role<br />
<strong>of</strong> PRC2 in regulating heart formation and energy metabolic homeostasis.<br />
3.13 Alternative splicing regulation during cardiac differentiation<br />
Verma S. K., Deshmukh V. & Kuyumcu-Martinez M. N.<br />
Department <strong>of</strong> Biochemistry and Molecular Biology, <strong>University</strong> <strong>of</strong> Texas Medical Branch, Galveston, TX 77555<br />
Alternative Splicing (AS) is a post-transcriptional process affecting ~95% <strong>of</strong> human genes and one <strong>of</strong> the major<br />
reason for proteome diversity. AS events are strictly regulated during heart development and embryonic stem<br />
cell differentiation into cardiomyocytes such that fetal is<strong>of</strong>orms are expressed only in embryonic but not in adult<br />
stages. Such regulation is essential to accommodate the functional differences between embryonic and adult<br />
heart. Even though AS is important for appropriate and specific is<strong>of</strong>orms <strong>of</strong> cardiac gene expression during<br />
development, the signals that regulate these transitions are largely unknown. Here, by using differentiation <strong>of</strong><br />
H9c2 rat embryonic heart cells into cardiomyocytes, we show that protein kinase C (PKC) activity is critical<br />
during cardiac development, and it regulates fetal AS events in embryonic hearts and also during cardiomyocyte<br />
differentiation via tight control <strong>of</strong> CUG-binding protein 1 (CUGBP1). Funding: AHA SDG-093022N, March <strong>of</strong><br />
Dimes Basil O'Connor starter scholar grant (#5 FY12-21) and UTMB start up funds to M.K-M<br />
3.14 MMAPPR: Mutation Mapping Analysis Pipeline for Pooled RNA-seq<br />
Jonathon T. Hill, Bradley Demarest, Brent Bisgrove, Bushra Gorsi, H. Joseph Yost<br />
Department <strong>of</strong> Neurobiology and Anatomy, <strong>University</strong> <strong>of</strong> Utah Molecular Medicine Program, Eccles Institute <strong>of</strong><br />
Human Genetics, Building 533, Room 3160, 15 North 2030 East, Salt Lake City, Utah 84112-5330, USA<br />
Forward genetic-screens in zebrafish are a vital tool for identifying novel genes essential for cardiovascular<br />
development or disease processes. However, one drawback <strong>of</strong> these screens is the labor-intensive and<br />
sometimes inconclusive process <strong>of</strong> mapping the causative mutation. Recent advances in high-throughput DNAsequencing<br />
technologies have made mapping mutations much more rapid in human populations. However, they<br />
have proven less effective in zebrafish because <strong>of</strong> its limited gene annotation, high polymorphism rate and the<br />
costs associated with performing these analyses on a large number <strong>of</strong> individuals. However, RNA-seq is<br />
commonly used on pools <strong>of</strong> zebrafish and shows strong potential for mapping recessive mutations. We propose<br />
an analysis pipeline for mapping mutations using RNA-seq, called MMAPPR (Mutation Mapping Analysis<br />
Pipeline for Pooled RNA-seq) to identify the causative mutation underlying an observed phenotype. MMAPPR<br />
(1) uses pooled samples without any knowledge <strong>of</strong> parental or individual genotypes, (2) accounts for the<br />
considerable amount <strong>of</strong> noise in RNA-seq datasets and (3) simultaneously identifies the region where the<br />
mutation lies and generate a list <strong>of</strong> putative coding region mutations in the linked segment that affect protein<br />
structure. We have validated our method on two known mutant lines (Nkx2.5 and Tbx1). We have also<br />
successfully mapped two novel cardiovascular mutants and are currently sequencing the identified candidate<br />
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genes. In addition to zebrafish, MMAPPR can be directly applied to other model organisms, such as D.<br />
melanogaster and C. elegans, where both forward genetic screens and pooled RNA-seq experiments are<br />
common. Supported by the NHLBI Bench-to-Bassinet Consortium Grant (U01HL0981)<br />
3.15 Capture and analysis <strong>of</strong> cardiac lineage-specific gene expression pr<strong>of</strong>iles<br />
Todd A. Townsend, H. Joseph Yost<br />
Department <strong>of</strong> Neurobiology and Anatomy, <strong>University</strong> <strong>of</strong> Utah Molecular Medicine Program, Eccles Institute <strong>of</strong><br />
Human Genetics, Building 533, Room 3160, 15 North 2030 East, Salt Lake City, Utah 84112-5330, USA.<br />
While several critical components <strong>of</strong> cardiovascular development have been elucidated, the gene expression<br />
pr<strong>of</strong>ile and signaling mechanisms that govern these processes is largely undetermined. Temporal and lineagespecific<br />
regulation <strong>of</strong> gene expression occurs throughout development and in response to distinct specific cell<br />
signaling events; however, current technology is constrained by the inability to effectively collect purified<br />
lineages at important developmental timepoints. To circumvent this problem we have developed transgenic<br />
zebrafish lines in which a biotin ligase recognition peptide (BLRP) is fused to a ribosomal protein (Rpl), and<br />
other lines in which lineage-specific expression <strong>of</strong> biotin ligase (BirA) in vitro biotinylates BLRP-Rpl. This BLRP-<br />
Rpl-BirA technology allows us to capture polysomes from specific cell lineages out <strong>of</strong> whole embryo lysates.<br />
With the lineage-specific polysomes in hand, we can then determine gene expression pr<strong>of</strong>iles from distinct<br />
tissues/cell lineages at specific developmental timepoints. Using a cardiac lineage specific (cmlc2/myl7)<br />
promoter to drive BirA expression, we have captured BLRP-biotin-tagged polysomes from developing cardiac<br />
lineages, and performed RNA-Seq and RT-PCR analysis. These gene expression pr<strong>of</strong>iles are being used to<br />
define cardiac-enriched transcripts. In addition, we are generating transgenic fish lines that specifically drive<br />
expression <strong>of</strong> BirA and BLRP-Rpl biotinylation in several specific cardiac lineages and in response to distinct<br />
cell signaling pathways. This powerful method will allow us to obtain genome-wide expression pr<strong>of</strong>iles within<br />
cardiac lineage at specific timepoints that are critical for heart development. Supported in part by AHA<br />
09POST2260423 and NHLBI 1F32HL114181 postdoctoral fellowships, and U01HL098179 (NHLBI Bench-to-<br />
Bassinet program).<br />
3.16 Identification <strong>of</strong> Intronic Sequence Responsible for Transcriptional Repression <strong>of</strong> etv2 in<br />
Myocardial Progenitors in the Zebrafish<br />
Marcus-Oliver Schupp and Ramani Ramchandran<br />
Developmental Vascular Biology Program, Department <strong>of</strong> Pediatrics, Children’s Research Institute and Medical<br />
College <strong>of</strong> Wisconsin, Milwaukee, Wisconsin, USA<br />
Gene regulatory networks governing the separation and maintanance <strong>of</strong> endothelial, endocardial and<br />
myocardial lineages from mesoderm remain poorly defined. During embryonic development <strong>of</strong> vertebrates, Etv2,<br />
an ETS family related transcription factor, is known to be indispensable for the initial specification and<br />
differentiation <strong>of</strong> endothelial cell lineages. Recently, FoxC1 factors were found to directly transactivate etv2 in<br />
zebrafish. Nevertheless, the transcriptional regulation <strong>of</strong> etv2 expression remains poorly understood.<br />
We applied reporter transgenics, co-expression analyses by fluorescent double in-situ-hybridisation, and<br />
morpholino-oligonucleotide mediated knockdown techniques to examine the cis-regulatory input <strong>of</strong> key<br />
transcription factors (TFs) into the etv2 gene locus in zebrafish. We found that reporter transgenes comprising<br />
regulatory regions upstream and downstream flanking the etv2 transcription start site robustly recapitulated<br />
endogenous etv2 expression in hematopoietic, endothelial and endocardial progenitors. Surprisingly, omission<br />
<strong>of</strong> intronic sequence resulted in ectopic activation <strong>of</strong> reporter transgene expression specifically in myocardial<br />
precursor cells. During heart cone stages (19-21 hpf), these cells co-expressed nkx2.5 and gata5 TFs that are<br />
fundamental to heart development. Co-expression with gata5 was seen already at 12 hpf, but not with nkx2.5.<br />
Knockdown <strong>of</strong> nkx2.5 activity enhanced expression <strong>of</strong> both intron-less and specific intron-harboring transgenes.<br />
In contrast, gata5 knockdown decreased expression <strong>of</strong> these transgenes. Further TFs are currently being<br />
investigated and presented at the conference. These studies suggest that we have identified an intronic cisregulatory<br />
element that is responsible in part for repression <strong>of</strong> the etv2 gene in myocardial progenitors. Our data<br />
also indicate a novel genetic mechanism linking endothelial/endocardial and myocardial cell lineages.<br />
3.17 Regulation <strong>of</strong> Heart Development by miR-17-92<br />
Stephanie B. Greene, Jun Wang, and James F. Martin<br />
Department <strong>of</strong> Molecular Physiology and Biophysics, Baylor College <strong>of</strong> Medicine, Houston, TX 77030<br />
Germline deletion <strong>of</strong> the microRNA (miRNA) cluster miR-17-92 in mouse models has previously been shown to<br />
104
esult in neonatal lethality due to impaired lung and B cell development, as well as ventricular septal defects. To<br />
determine the roles <strong>of</strong> miR-17-92 exclusively in heart development, we used cardiac-specific Cre lines with both<br />
conditional over-expression and knock-out alleles. Here we show that both over-expression and deletion <strong>of</strong> miR-<br />
17-92 in the cardiac lineage lead to ventricular septal defects and other cardiomyopathies.<br />
We are currently analyzing the potential for the miR-17-92 cluster to regulate mRNA expression genome-wide<br />
by performing RNA-Immunoprecipitation microarrays (“RIP-chip”) <strong>of</strong> Argonaut 2-bound RNA purified from<br />
mutant and control tissues. This method is useful to purify target mRNAs specifically bound to the miRNA-RISC<br />
(Ago2) machinery. Analysis <strong>of</strong> data from the RIP-chip has revealed several candidate target genes that overlap<br />
with miRNA target predictions. We are currently validating several <strong>of</strong> these predicted targets and determining<br />
how they interact with miR-17-92. These include several members <strong>of</strong> the Wnt signaling pathway. Accordingly,<br />
when miR-17-92 is conditionally knocked out, we observe decreased active beta-catenin.<br />
3.18 Discovering small molecules that promote cardiomyocyte generation by modulating Wnt signaling<br />
Terri T. Ni 1,2,6+, Eric J. Rellinger 2+ , Amrita Mukherjee 2,4 , Lauren Stephens 2 , Cutris A Thorne 4 ,<br />
Kwangho Kim 5,6 , Jiangyong Hu 2,4 , Shuying Xie 1 , Ethan Lee 4,6 , Larry Marnett 3,6 , Antonis K. Hatzopoulos 2,4 , Tao<br />
P. Zhong 1,2,4,6<br />
1 School <strong>of</strong> Life Sciences, Fudan <strong>University</strong>, Shanghai 20043, China; 2 Department <strong>of</strong> Medicine, 3 Department <strong>of</strong><br />
Pharmacology, 4 Department <strong>of</strong> Cell & Developmental Biology, 5 Department <strong>of</strong> Chemistry, 6 Vanderbilt Institute <strong>of</strong><br />
Chemical Biology, Vanderbilt <strong>University</strong> School <strong>of</strong> Medicine, Nashville, TN 37203, USA.<br />
We have developed a robust in vivo small molecule screen that modulates heart size and cardiomyocyte<br />
generation in zebrafish. Three structurally-related compounds (Cardionogen-1 to -3) identified from our screen<br />
enlarge the size <strong>of</strong> the developing heart via myocardial hyperplasia. Increased cardiomyocyte number in<br />
Cardionogen-treated embryos is due to expansion <strong>of</strong> cardiac progenitor cells. In zebrafish embryos and murine<br />
embryonic stem (ES) cells, Cardionogen treatment promotes cardiogenesis during and after gastrulation,<br />
whereas inhibits heart formation before gastrulation. Cardionogen-induced effects can be antagonized by<br />
increasing Wnt/β-catenin signaling activity. We demonstrate that Cardionogen inhibits Wnt/β-catenin-dependent<br />
transcription in murine ES cells and zebrafish embryos. Cardionogen can rescue Wnt8-induced cardiomyocyte<br />
deficiency and heart-specific phenotypes during development. These findings demonstrate that in vivo small<br />
molecule screens targeted on heart size can discover compounds with cardiomyogenic effects and identify<br />
underlying target pathways. *The paper has been published in Chemistry & Biology. 2011 Dec 23;18(12):16<br />
3.19 ETS2 and Mesp1 transdifferentiate human dermal fibroblasts into cardiac progenitors<br />
Jose F. Islas* 1,2 , Yu Liu 3 , Kuo-Chan Weng* 1,2 , Allan Prejusa 1 , Tsuey-Ming Chen 1 , Dinakar Iyer 4 , Vladimir<br />
Potaman 2 and Robert J. Schwartz 1,3<br />
1 Texas Heart Institute, Texas Medical Center, Houston, 2 The Institute <strong>of</strong> Bioscience and Technology, Texas<br />
A&M Health Science Center Houston, 3 Department <strong>of</strong> Biology and Biochemistry, <strong>University</strong> <strong>of</strong> Houston,<br />
4 Department <strong>of</strong> Medicine Baylor College <strong>of</strong> Medicine, Texas Medical Center, Houston Texas 77030<br />
Heart failure is one <strong>of</strong> the leading causes <strong>of</strong> death in industrialized countries. Due to the shortage <strong>of</strong> available<br />
donors for transplants, alternative therapies are being developed which are based on usage <strong>of</strong> embryonic stem<br />
(ES) and induced pluripotency (IPS) cells. Usage <strong>of</strong> IPS cells in cardiac cell replacement therapy is<br />
advantageous because they circumvent inmuno-rejection problems, since they can be produce directly from the<br />
patient’s own cells. Unfortunately, current techniques <strong>of</strong> IPS cell production, still lead to low yields <strong>of</strong> full<br />
undifferentiated cells and even lower yields in sequential differentiation procedures.<br />
Our study centers on the design <strong>of</strong> an effective manner to convert Normal Human Dermal Fibroblasts into<br />
cardiac progenitors, through the usage <strong>of</strong> transcription factors v-ets erythroblastosis virus E26 oncogene<br />
homolog 2 (ETS2) and mesoderm posterior 1 homolog (Mesp1). The first, ETS2 being a known oncogene,<br />
which potentially initiates a proliferative activity by c-Myc control and the second MesP1 an early mesoderm<br />
marker implicated in cardiac formation. From the developmental perspective, ETS2/Mesp1 treated cells have<br />
significant cardiomyocyte potential. They express an cardiac progenitor program represented by NKX2.5,<br />
MEF2C TBX5, GATA4 and ISL1, contractility and electrical coupling represented by MHC3, MLC2, TNT, CX43,<br />
CX45, as well as cell surface mesoderm markers Flk1/KDR and PDGFRa required in an obligatory fashion<br />
during cardiomyocyte development.<br />
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3.20 Cell surface chemoproteomics for capturing states <strong>of</strong> cardiac differentiation from pluripotent stem<br />
cells<br />
Subarna Bhattacharya, Sandra Chuppa, Olena Wiedemeier and Rebekah L. Gundry<br />
Department <strong>of</strong> Biochemistry and Biotechnology and Bioengineering Center, Medical College <strong>of</strong> Wisconsin,<br />
8701 Watertown Plank Road, Milwaukee, WI 53226.<br />
The ability to transplant cardiomyocytes to effect myocardial repair and remuscularization is a major unfulfilled<br />
goal <strong>of</strong> regenerative medicine. To address this, the current study aims to develop a non-transgene-based<br />
method to isolate transplantable numbers (tens <strong>of</strong> millions) <strong>of</strong> purified, functionally defined cardiomyocytes using<br />
immunophenotyping, a strategy well-defined for isolating therapeutically viable hematopoietic stem cells. To<br />
identify surface marker panels unique to cardiomyogenic subtypes (e.g. atrial, ventricular), we utilize a state-<strong>of</strong>the-art<br />
quantitative chemoproteomic approach that does not rely on the availability or specificity <strong>of</strong> antibodies –<br />
high mass accuracy mass spectrometry (MS) coupled with extracellular protein domain capturing – a strategy<br />
that enables epitope discovery and quantitation. This strategy is applied to a well-defined mouse embryoid<br />
body-based model <strong>of</strong> cardiomyogenesis to reveal cell surface proteins that quantitatively change during<br />
mesoderm commitment and cardiac differentiation. Subsequently, antibody-based cell sorting is used to isolate<br />
cardiomyocyte subtypes based on surface epitope expression, followed by molecular, electrophysiological, and<br />
functional assessment. To date, this strategy has identified >600 cell surface proteins among the pluripotent and<br />
cardiomyocyte populations studied. From these, >30 proteins are signficantly enriched in the cardiomyocyte<br />
population. Of these, several candidate proteins were chosen for further analysis by flow cytometry,<br />
immunoblotting, RT-PCR, and whole mount immunostaining to determine when and where these markers<br />
appear during various stages <strong>of</strong> differentiation and in vivo development. In summary, these results indicate that<br />
cell surface proteins display distinct expression patterns during cardiomyogenesis that may enable the sorting <strong>of</strong><br />
distinct subpopulations <strong>of</strong> cardiomyocytes for basic science studies and therapeutic applications.<br />
3.21 Gata5 efficiently directs formation <strong>of</strong> cardiac progenitors with atrial-like potential from murine<br />
embryonic stem cells<br />
Harma Khachig Turbendian MD 1 , Miriam Gordillo PhD 1 , Jia Lu PhD 2 , Guoxin Kang PhD 2 , Ting-Chun Liu MS 1 ,<br />
Glenn I. Fishman MD 2 , Todd Evans PhD 2<br />
1 2<br />
Department <strong>of</strong> Surgery, Weill Cornell Medical College, New York, NY; Leon H. Charney Division <strong>of</strong> Cardiology,<br />
New York <strong>University</strong> School <strong>of</strong> Medicine, New York, NY<br />
Many <strong>of</strong> the transcription factors controlling cardiogenesis are known and comprise a molecular network<br />
essential for normal heart growth, morphogenesis, and function. However, there is less known about the<br />
mechanisms that regulate the development <strong>of</strong> specific cardiac lineages from embryonic stem cells. The<br />
transcription factors GATA4/5/6 have been shown to function redundantly in the regulation <strong>of</strong> cardiac<br />
development. In vivo studies suggest that Gata5 may control a genetic program involved in the development<br />
and specification <strong>of</strong> cardiomyocyte fate, including septal development and valvulogenesis. We have developed<br />
a system for conditional expression <strong>of</strong> Gata5 in murine embryonic stem cells (mES), and tested whether Gata5<br />
can direct specification <strong>of</strong> cardiomyocyte fate, evaluated by qPCR, immunohistochemisty, flow cytometry, and<br />
electrophysiology. We show that induction <strong>of</strong> Gata5 expression at day 4 <strong>of</strong> embryoid body (EB) development in<br />
serum-free conditions results in derivatives that demonstrate extensive contractility. Typically, at least 50% <strong>of</strong><br />
the cells within Gata5-induced EB derivatives express cardiac troponin T (cTnt) by day 12. A significant<br />
proportion <strong>of</strong> these cells represent immature cardiac precursors as demonstrated by co-expression <strong>of</strong> cTnt and<br />
smooth muscle actin alpha. Additionally, expression <strong>of</strong> Gata5 within this defined window directs development <strong>of</strong><br />
atrial-like progenitors as identified by their protein and electrophysiologic pr<strong>of</strong>ile. These cells express the atrial<br />
is<strong>of</strong>orm <strong>of</strong> myosin regulatory light chain 2, show heterogeneous action potential morphology, and display<br />
responsiveness to adrenergic and muscarinic stimulation. These findings suggest that Gata5 functions<br />
downstream <strong>of</strong> known procardiac signaling programs to direct specification <strong>of</strong> progenitors with atrial-like<br />
potential from mES.<br />
3.22 Salls Promote Cardiac Progenitor Differentiation to Specify Cardiac Cell Lineages<br />
Morita Y. 1.2 , Tsukahara Y. 2 , Anderson P. 3 , Kurokawa J. 4 , Sugizaki H. 1 , Nishinakamura R. 5 , Kwon C. 3 , Koshiba-<br />
Tkaeuchi 1.2 & Takeuchi JK. 1.2.6<br />
1 IMCB, the <strong>University</strong> <strong>of</strong> Tokyo, 2 Guraduate School <strong>of</strong> Sciences, the <strong>University</strong> <strong>of</strong> Tokyo, 3 Cardiology, Johns<br />
Hopkins <strong>University</strong>, 4 Tokyo Medical and Dental <strong>University</strong>, 5 CARD, Kumamoto <strong>University</strong>, 6 JST PRESTO<br />
The fundamental approaches what we should progress in mammal cardiac program research will be to identify<br />
106
defined factors for induced cardiac stem cells/progenitor cells (iCPCs), and to generate the methods for<br />
induction <strong>of</strong> cardiac cells with high efficiency through their progenitor cell-fate in vivo/in vitro systems.<br />
The knowledge gained from the developmental studies led to the recent breakthrough discoveries <strong>of</strong> 3 defined<br />
factors, whose co-overexpression is sufficient to “direct (re)-program” non-cardiac cells to convert into<br />
cardiomyocytes. Based on these findings, we have identified Sall genes as the new candidate markers to CPCs<br />
from 15 genes that were highly enriched in Islet/Flk1(+)-CPCs in mice embryos. Sall-EGFP knock-in ES cells<br />
and sorted Sall(+) cells from mouse embryos significantly differentiated into ventricular-like cardiomyocytes,<br />
conduction cells including SAN, smooth muscle and endothelial cells, indicating Sall(+) cells position upstream<br />
<strong>of</strong> Islet1/Nkx2-5-WT1-CPCs. Surprisingly, mostly Sall-EGFP(+) colonies differentiated into beating<br />
cardiomyocyte (80%) with cardiac Troponin (>90%), whereas EGFP(-) cells differentiated into cardiomyocytes<br />
with low efficiency (
3.25 Combinatorial Regulation <strong>of</strong> Cardiac Gene Expression by ETS and Mesp1 transcription factors<br />
Kuo-Chan Weng 1,2 , Yu Liu 3 , Litao Yang 3 , Ashley Benham 3 , Jose F. Islas 1,2 , Vladimir N. Potaman 2 , Robert J.<br />
Schwartz 2,3<br />
1 The Institute <strong>of</strong> Bioscience and Technology, Texas A&M Health Science Center, Houston, Texas 77030<br />
2 Texas Heart Institute, Texas Medical Center, Houston, Texas 77030<br />
3 Department <strong>of</strong> Biology and Biochemistry, <strong>University</strong> <strong>of</strong> Houston, Texas 77204<br />
Mesp1 plays a key role in cardiac lineage commitment contributing to the development <strong>of</strong> both the first and<br />
second heart fields. It is a bHLH transcription factor that binds to the conserved E-box sequences in DNA.<br />
Current knowledge <strong>of</strong> the downstream genes and protein partners <strong>of</strong> Mesp1 is limited. Among potential Mesp1<br />
partners are the ETS family transcription factors which contain conserved DNA-binding ETS domains and are<br />
also essential for cardiac development. Thus, transcripts <strong>of</strong> several ETS factors are abundant in the early<br />
embryonic heart, whereas ETS2-knockout ES cells fail to beat and do not express cardiac markers. We have<br />
started the Chip-seq analyses <strong>of</strong> the mouse embryoid bodies using antibodies to endogenous Mesp1 and ETS<br />
family proteins to determine global distribution <strong>of</strong> their binding elements. Gene Ontology analysis relates an<br />
abundance <strong>of</strong> determined closely spaced E-box/ETS binding sites to the upstream sequences <strong>of</strong> the genes<br />
involved in cardiac development. Initial tests <strong>of</strong> such elements by the Luciferase assay have shown that pairwise<br />
binding <strong>of</strong> Mesp1 and several ETS factors to the upstream sequence <strong>of</strong> the early cardiac gene Nkx2-5<br />
results in a synergistic transcriptional activation. Co-immunoprecipitation has shown physical association<br />
between several ETS transcription factors and Mesp1. Thus, our studies indicate that physical and functional<br />
interactions between Mesp1 and ETS transcription factors may play important roles in cardiac development.<br />
3.26 Dissecting Tbx5 function: roles for the novel Tbx5b paralog in cardiogenesis<br />
Molly Ahrens, Troy Camarata, Brandon Holtrup, Hans-Georg Simon<br />
Northwestern <strong>University</strong> and Children's Memorial Research Center<br />
Congenital Heart Disease (CHD) is the most frequent birth defect, and mutations in transcription factors,<br />
including T-box proteins, are causative in many <strong>of</strong> these CHDs. Mutations in one family member, TBX5, results<br />
in the autosomal dominant condition Holt-Oram Syndrome (HOS), characterized by heart malformations and<br />
limb deformities. Tbx5 is evolutionarily conserved, and zebrafish deficient for Tbx5 mimic many aspects <strong>of</strong> the<br />
HOS phenotype. However, how Tbx5 downstream activities control cellular functions and tissue morphogenesis<br />
has remained elusive. Recently, our lab identified a novel Tbx5 paralog (Tbx5b) in zebrafish. My preliminary<br />
data demonstrate that Tbx5a (previously Tbx5) and Tbx5b have distinct expression pr<strong>of</strong>iles throughout most <strong>of</strong><br />
heart development, suggesting that these paralogs have evolved specialized functions that constitute the single<br />
Tbx5 <strong>of</strong> other vertebrates. This natural model <strong>of</strong> functional variation provides the unique opportunity to gain a<br />
deeper under standing <strong>of</strong> the distinct developmental processes <strong>of</strong> cardiogenesis. Thus, I hypothesize that Tbx5b<br />
has specialized regulatory functions at the cellular level that control morphogenesis into cardiac chambers and<br />
outflow tract, including valves. Determining these cellular activities downstream <strong>of</strong> Tbx5b is critical for extending<br />
our knowledge <strong>of</strong> the full range <strong>of</strong> Tbx5 functions in mammals and thus developing novel treatments for CHD.<br />
3.27 Tbx1 interacts with the BAF chromatin remodeling complex.<br />
FG. Fulcoli 1 , L. Chen 2 , S. Martucciello 3 , EA. Illingworth 1,3 , A. Baldini 1<br />
1 2 3<br />
Institute <strong>of</strong> Genetics and Biophysics, CNR, Naples, Italy. <strong>University</strong> <strong>of</strong>, Houston, Houston, USA. <strong>University</strong> <strong>of</strong><br />
Salerno, Salerno, Italy.<br />
The T-Box transcription factor Tbx1 is required for a number <strong>of</strong> developmental processes, including the<br />
regulation <strong>of</strong> proliferation, differentiation and, possibly migration <strong>of</strong> cardiac progenitors <strong>of</strong> the second heart field<br />
(SHF). However, the mechanisms by which Tbx1 exerts its transcriptional functions are not well understood.<br />
Here we tested interactions between Tbx1 and chromatin remodelers and histone modifiers and asked how<br />
these interactions affect the transcription <strong>of</strong> target genes. We have identified a novel target <strong>of</strong> Tbx1, Wnt5a, a<br />
gene encoding for a ligand <strong>of</strong> the non canonical Wnt pathway. Wnt5a is expressed in the SHF, and is required<br />
for cardiac outflow tract development. Using genetic crosses, we demonstrated that Wnt5a and Tbx1 interact<br />
genetically. Wnt5a harbors two Tbx1-responding enhancers. We used these enhancers to explore the<br />
mechanisms <strong>of</strong> Tbx1 function. We established that Tbx1 interacts directly with Baf60a, a subunit <strong>of</strong> the BAF<br />
chromatin remodeling complex. Increased dosage <strong>of</strong> Tbx1 enhances the occupation <strong>of</strong> Baf60a onto the Wnt5a<br />
gene in vivo and in tissue culture. Knock-down <strong>of</strong> Baf60a abolishes the ability <strong>of</strong> Tbx1 to regulate the Wnt5a<br />
gene as well as other targets. Furthermore, we found a physical interaction with the histone methyltransferase<br />
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Setd7 and the enrichment <strong>of</strong> H3K4 monomethylation status on the target loci after increased dosage <strong>of</strong> Tbx1.<br />
Our data indicate the importance <strong>of</strong> Baf60a for Tbx1-induced regulation <strong>of</strong> Wnt5a and perhaps other target<br />
genes. Hence it is reasonable to speculate that this BAF subunit is a key c<strong>of</strong>actor for Tbx1 function in cardiac<br />
progenitors.<br />
3.28 ChIP-seq identification <strong>of</strong> downstream targets <strong>of</strong> Nkx2.5 during heart development<br />
Laurent Dupays 1 , Robert Wilson 1 , Surendra Kotecha 1 , Norma Towers 1 , Richard Harvey 2 and Timothy Mohun 1<br />
1<br />
Division <strong>of</strong> Developmental Biology, MRC-National Institute for Medical Research, Mill Hill, London, UK.<br />
2<br />
Victor Chang Cardiac Research Institute and Faculties <strong>of</strong> Medicine and Life Science, <strong>University</strong> <strong>of</strong> New South<br />
Wales, Sydney, Australia.<br />
Nkx2.5 function has been shown to be essential at various stages <strong>of</strong> mouse heart development, in addition to<br />
playing an important role in physiology <strong>of</strong> the newborn heart. Moreover, human NKX2.5 mutations have been<br />
suggested as the underlying cause <strong>of</strong> a variety <strong>of</strong> congenital heart diseases. Despite extensive analyses, the<br />
downstream effectors mediating Nkx2.5 function during heart development remain largely unknown. In order to<br />
identify mediators <strong>of</strong> Nkx2.5 function in vivo we used chromatin immunoprecipitation followed by massive<br />
parallel sequencing (ChIP-seq) in the E11.5 mouse heart. We were able to map several thousands <strong>of</strong> in vivo<br />
binding sites for Nkx2.5 in the mouse genome at that stage. Interestingly, a large number <strong>of</strong> the regions bound<br />
by Nkx2.5 have already been shown to be bound by the enhancer associated protein p300. Thirty-seven <strong>of</strong> the<br />
regions binding both Nkx2.5 and p300 have been previously shown to have cardiac-specific enhancer activity in<br />
vivo in transgenic mouse reporter assays suggesting that these proteins act together to regulate a subset <strong>of</strong><br />
cardiac genes. To discriminate between functional and non-functional binding, we compared this data set with<br />
changes in gene expression, using expression array analysis <strong>of</strong> a hypomorphic model <strong>of</strong> Nkx2.5 expression.<br />
With this combined approach, we identified 116 genes dysregulated in the Nkx2.5 hypomorph at E11.5 that<br />
contain a Nkx2.5 binding site, strongly suggesting that they are direct targets <strong>of</strong> Nkx2.5. Surprisingly, more than<br />
30% <strong>of</strong> these direct targets have a function related to the regulation <strong>of</strong> energy metabolism, suggesting that<br />
Nkx2.5 acts as a key regulator <strong>of</strong> the metabolic processes during cardiac development.<br />
3.29 A meta-analysis <strong>of</strong> global gene expression and interaction in heart development <strong>of</strong> mouse<br />
Linglin Xie 1 and Kurt Zhang 2<br />
Department <strong>of</strong> Biochemistry 1 , Department <strong>of</strong> Pathology and Bioinformatics Core 2<br />
School <strong>of</strong> Medicine and Health Sciences, <strong>University</strong> <strong>of</strong> North Dakota<br />
Heart development is a complicated biological process that involves the activations and interactions <strong>of</strong><br />
thousands <strong>of</strong> genes that form a gene regulatory network. To unravel this gene network is highly challenging<br />
because we have limited knowledge about gene functions and gene-gene interaction and it is computationally<br />
difficult to infer from the high-dimensional genomic data. The fast advancement <strong>of</strong> high throughput<br />
biotechnology is generating a wealth <strong>of</strong> genome-wide information regarding gene expression, DNA binding,<br />
DNA and RNA sequences, and epigenetic effects that provides an unprecedented opportunity to understand<br />
gene functions and interactions during heart development. In this project, we have integrated gene expression<br />
pr<strong>of</strong>iles from multiple developmental stages and ChIP-Seq data for a few important transcriptional factors that<br />
are involved in heart development in mouse. A meta-analysis was conducted to illustrate gene differentially<br />
expression at different developmental stages and to infer the gene interactions and activated pathways for heart<br />
development.<br />
Section 4: Neural Crest and Vascular Development<br />
S4.1 Early neural crest-restricted Noggin over-expression results in congenital crani<strong>of</strong>acial and<br />
cardiovascular defects<br />
Zachary Neeb, Paige Snider and Simon J. Conway<br />
Program in Developmental Biology and Neonatal Medicine, Herman B Wells Center for Pediatric Research,<br />
Indiana <strong>University</strong> School <strong>of</strong> Medicine, Indianapolis, IN 46202.<br />
Noggin is a secreted BMP antagonist that binds BMP ligands and prevents their activation <strong>of</strong> receptors. Noggin<br />
(in concert with Wnts, FGFs, Delta/Notch) plays a key upstream role in neural crest cell (NCC) induction by<br />
generating a BMP morphogenic gradient that activates a cascade <strong>of</strong> transcription factors culminating in the<br />
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ultimate steps <strong>of</strong> NCC differentiation. NCC derivatives are absolutely essential for cardiac outflow tract (OFT)<br />
remodeling, specifically during OFT septation and patterning <strong>of</strong> the great vessels that exit the heart.<br />
Using Cre/loxP, constitutive Noggin expression was induced within NCC via Pax3 Cre (very early within neural<br />
tube), Wnt1-Cre (early within neural tube) or Peri-Cre (post-migratory) drivers. Consistent with its in vitro role in<br />
negatively regulating BMP signaling, Westerns confirmed Noggin overexpression significantly suppressed<br />
phosphorylation levels <strong>of</strong> Smad1/5/8 but left Smad2/3 unaffected in vivo. Moreover, Wnt1-Cre and Pax3 Cre -<br />
mediated suppression <strong>of</strong> BMP signaling resulted in subsequent loss <strong>of</strong> NCC-derived crani<strong>of</strong>acial, pharyngeal<br />
and OFT cushion tissues. Increased cell death was observed in pharyngeal arch NCC and DRG (although cell<br />
proliferation was unaltered), but not within the OFT itself. Lineage mapping demonstrated that NCC emigration<br />
was not affected in either Pax3 Cre ;Noggin or Wnt1-Cre;Noggin mutants, but that subsequent colonization <strong>of</strong> the<br />
OFT was significantly reduced in Wnt1-Cre;Noggin and completely absent in Pax3 Cre ;Noggin mutants. Further,<br />
although Wnt1-Cre;Noggin mutants are viable until birth, Pax3 Cre ;Noggin mutants all die by E14. Conversely,<br />
Peri-Cre mediated suppression <strong>of</strong> BMP signaling in post-migratory NCC had no effect and mutants are viable at<br />
birth. Taken together, these data show that conditional BMP inhibition within the NCC may exhibit different<br />
effects dependent upon timing and that tightly regulated TGFβ superfamily signaling plays an essential role<br />
during crani<strong>of</strong>acial and cardiac NCC survival in vivo.<br />
S4.2 The Role <strong>of</strong> Fibronectin - Integrin Signaling in Pharyngeal Microenvironment in Modulation <strong>of</strong><br />
Cardiac Neural Crest Cell Development<br />
Dongying Chen and Sophie Astr<strong>of</strong><br />
Thomas Jefferson <strong>University</strong>, Philadelphia, USA<br />
Patterning <strong>of</strong> Pharyngeal Arch Arteries (PAAs) is a critical process in embryonic cardiovascular development.<br />
Alterations that perturb this process cause cardiovascular abnormalities such as human DiGeorge Syndrome.<br />
Our lab previously discovered defective development <strong>of</strong> CNCCs in global fibronectin (FN)-null or integrin a5-null<br />
mutants. In order to determine the roles <strong>of</strong> FN synthesized by the pharyngeal microenvironment during CNCC<br />
development, we conditionally ablated FN or the α5 subunit <strong>of</strong> its integrin receptor using Isl1 Cre strain <strong>of</strong> mice.<br />
We found that conditional inactivation <strong>of</strong> FN or integrinα5 using Isl1 Cre cause embryonic/neonatal lethality. Both<br />
<strong>of</strong> these conditional mutants display various cardiovascular and glandular anomalies associated with defective<br />
PAA and CNCC development. Moreover, histological analyses reveal defective development <strong>of</strong> ventricular<br />
myocardium and muscular ventricular septum, phenotypes dependent on the proper development <strong>of</strong> the<br />
mesodermal cardiac progenitors. Taken together, these phenotypes suggest that FN-integrin α5 signaling to<br />
distinct tissues comprising pharyngeal microenvironment play critical roles in PAA development by modulating<br />
interactions between CNCCs and non-CNCCs. To understand the basis <strong>of</strong> these phenotypes, we have<br />
investigated the contribution <strong>of</strong> CNCC to PAA formation/remodeling and found that FN promotes survival <strong>of</strong><br />
CNCCs and cardiac progenitors. In addition, FN synthesized by pharyngeal tissues regulates CNCC navigation.<br />
We are investigating possible roles <strong>of</strong> FN and integrin α5-regulated signaling in regulation <strong>of</strong> morphogens,<br />
guidance molecules and growth factor signaling to tissues comprising the pharyngeal microenvironment. Taken<br />
together, our studies provide important insights into how tissue microenvironment regulates morphogenesis <strong>of</strong><br />
complex organs from diverse populations <strong>of</strong> progenitors.<br />
S4.3 Slit3-Robo1/2 signalling controls cardiac innervation and ventricular septum development by<br />
regulating neural crest cell survival<br />
Mathilda T.M. Mommersteeg 1 , William D. Andrews 1 , Alain Chédotal 2 , Vincent M. Christ<strong>of</strong>fels 3 , John G.<br />
Parnavelas 1<br />
1<br />
Department <strong>of</strong> Cell and Developmental Biology, <strong>University</strong> College London, London, UK<br />
2<br />
Institut National de la Santé et de la Recherche Médicale, UMR S968, Institut de la Vision, Paris, France<br />
3<br />
Heart Failure Research Center, Department <strong>of</strong> Anatomy, Embryology and Physiology, Amsterdam, The<br />
Netherlands<br />
The Slit-Robo signalling pathway has been shown to have pleiotropic effects in Drosophila heart development,<br />
however, its involvement in mammalian heart formation is yet largely unknown. Here, we analysed the role <strong>of</strong><br />
this signalling pathway during murine heart development. We observed extensive expression <strong>of</strong> both Robo1 and<br />
Robo2 receptors and their ligands, Slit2 and Slit3, in and around the developing heart. Analysis <strong>of</strong> mice lacking<br />
Robo1 revealed membranous ventricular septum defects and decreased cardiac innervation, while animals<br />
lacking Robo2 did not display such defects. However, the combined absence <strong>of</strong> both Robo1 and Robo2 caused<br />
increased incidence and severity <strong>of</strong> membranous ventricular septum defects. Mice lacking the Slit1 and Slit2<br />
ligands did not reveal any abnormalities, but Slit3 mutants recapitulated the membranous ventricular septum<br />
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defect. Interestingly, the density <strong>of</strong> cardiac innervation was increased in Slit3 mutants. The combined absence<br />
<strong>of</strong> the membranous ventricular septum and innervation defects suggested a defect in cardiac neural crest<br />
contribution. Detailed cell counts showed a reduction in neural crest cell contribution to the outflow tract. Further<br />
analyses indicated increased apoptosis in the neural crest, just before entering the heart, suggesting that cell<br />
death underlies its reduced contribution to the heart. Our data indicate a novel role for Slit3-Robo1/2 interaction<br />
in cardiac neural crest survival and, thereby, in the formation <strong>of</strong> the membranous ventricular septum and cardiac<br />
innervation.<br />
S4.4 The neural crest contributes to coronary artery smooth muscle formation through endothelin<br />
signaling<br />
Yuichiro Arima*, Sachiko Miyagawa-Tomita**, Kazuhiro Maeda**, Koichi Nishiyama*, Rieko Asai*, Ki-Sung<br />
Kim*, Yasunobu Uchijima*, Hisao Ogawa***, Yukiko Kurihara* and Hiroki Kurihara*<br />
*Department <strong>of</strong> Physiological Chemistry and Metabolism, Graduate School <strong>of</strong> Medicine, <strong>University</strong> <strong>of</strong> Tokyo,<br />
Tokyo, Japan. **Department <strong>of</strong> Pediatric Cardiology, Tokyo Women’s Medical <strong>University</strong>, Tokyo, Japan.<br />
*** Department <strong>of</strong> Cardiovascular Medicine, Faculty <strong>of</strong> Life Sciences, Kumamoto <strong>University</strong>, Kumamoto, Japan.<br />
Endothelin-1 (ET1) /Endothelin A receptor (ETAR) axis has important roles in regulating cardiovascular<br />
homeostasis and organ development. We have previously reported that ET1 and ETAR knockout mice display<br />
crani<strong>of</strong>acial defects and aortic arch malformations. We also found their expressions in the developing coronary<br />
artery smooth muscle cells (CASMCs), however their roles in coronary artery development had been unknown.<br />
Here we show that ET1/ETAR axis insufficiency disturbs coronary artery development. Fetal coronary<br />
angiography revealed some septal branches were abnormally enlarged in both ET-1 and ETAR KO mice at<br />
embryonic day 17.5 (E17.5) and the branching pattern <strong>of</strong> the septal branch was also altered in both KO mice.<br />
Immunohistochemical analysis revealed partial lack <strong>of</strong> CASMCs in KO mice and serial analysis showed these<br />
malformations were occurred at the remodeling stage <strong>of</strong> coronary arteries. Each phenotype appeared in the<br />
septal branch-specific manner, then we hypothesized these segment specificities stood on the variety <strong>of</strong> cell<br />
sources <strong>of</strong> CASMCs. Fate mapping using Wnt1-Cre mice suggested that lacked CASMCs were specifically<br />
derived from the neural crest cells. To analyze the contribution <strong>of</strong> neural crest cells to CASMCs in detail, we<br />
used quail-chick chimera technique and neural crest ablation models. Quail-chick chimera with a particular<br />
region <strong>of</strong> the neural crest recaptured the preferential distribution pattern to the interventricular septum region.<br />
Chick ablation models also showed coronary artery malformations similar to those in ET-1 and ETAR KO mice.<br />
These results indicate novel contribution <strong>of</strong> the neural crest to coronary artery formation partly through<br />
endothelin signaling.<br />
S4.5 CASTOR directly regulates a novel Egfl7/RhoA pathway to promote blood vessel development and<br />
morphogenesis<br />
Marta Szmacinski, Kathleen Christine, Nirav Amin, Joan Taylor, Frank L. Conlon<br />
The <strong>University</strong> <strong>of</strong> North Carolina, Chapel Hill<br />
The formation <strong>of</strong> the vascular system is essential for embryonic development and homeostasis. Consistent with<br />
recent genome wide association studies implicating a genetic link between the transcription factor CASTOR<br />
(CST) and high blood pressure and hypertension, we demonstrate here that CST has an evolutionarily<br />
conserved function in blood vessel formation; in the absence <strong>of</strong> CST, Xenopus embryos fail to develop a fully<br />
branched and lumenized vascular system and human endothelial cells (HUVECs) lacking CST display dramatic<br />
defects in adhesion/contractility and cell division. Using chromatin immunoprecipitation (ChIP), we go on to<br />
identify Epidermal growth factor-like domain 7 (Egfl7) as a direct transcriptional target <strong>of</strong> CST. We demonstrate<br />
that CST is required for expression <strong>of</strong> Egfl7, that CST is endogenously bound to the Egfl7 locus in the<br />
developing embryo, and that depletion <strong>of</strong> EGFL7 phenocopies CST depletion in whole embryos and in<br />
HUVECs. We further show that the adhesio n/contractility abnormalities due to depletion <strong>of</strong> CST in HUVECs can<br />
be rescued by the reintroduction <strong>of</strong> EGFL7. Critically, we have demonstrated that CST and EGFL7 modulate<br />
endothelial cell shape and contractility through the small GTPase RhoA. Collectively, these data support a<br />
mechanism whereby CST directly regulates its transcriptional target Egfl7 to promote RhoA-mediated<br />
endothelial cell shape and adhesion changes to establish the vasculature and hence, these studies provide<br />
insight into the cellular and molecular basis for vascular disease associated with the loss <strong>of</strong> CST.<br />
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S4.6 Nephronectin regulates axial vein morphogenesis in zebrafish<br />
Chinmoy Patra* 1 , Filomena Ricciardi 1 , Iva Nikolic 2 , Mirko HH Schmidt 2 , Benno Jungblut 1 , Felix B. Engel 1<br />
Max-Planck-Institute for Heart and Lung Research<br />
Angiogenesis is the development <strong>of</strong> new vessels from pre-existing vessels. This is a critical morphological event<br />
both in organ development as well as in diseases. Like in other vertebrates, in zebrafish vessels form a complex<br />
network in order to fulfill tissue oxygen demands. Development <strong>of</strong> complex vascular networks is dependent on<br />
the directional migration <strong>of</strong> groups <strong>of</strong> endothelial cells, which is called angiogenic sprouting. Here we have<br />
demonstrated that in zebrafish the extracellular matrix protein, nephronectin, is transiently expressed in the<br />
caudal vein plexus forming region at the time <strong>of</strong> caudal vein sprouting at around 30 hours post fertilization (hpf).<br />
Morpholino-mediated nephronectin depletion resulted in the malformation <strong>of</strong> the caudal vein plexus and the<br />
ventral vein and in the frequent loss <strong>of</strong> inter-segmental veins. Time-lapse analysis from 28 hpf to 40 hpf<br />
indicated a decreased in the frequency <strong>of</strong> caudal vein sprout formation in nephronectin morphants. In addition,<br />
existing sprouting appeared multi-directional indicating a navigation problem. Biochemical analysis<br />
demonstrated that nephronectin is able to bind to the integrin αV/β3 heterodimer. Importantly, integrin αV and<br />
nephronectin expression overlapped in the region <strong>of</strong> the caudal vein plexus. Moreover, morpholino-mediated<br />
integrin αV knockdown in zebrafish phenocopied nephronectin depletion. Taken together, our data indicate that<br />
nephronectin regulates directional sprouting <strong>of</strong> the axial vein in zebrafish, which might be via integrin αV.<br />
4.7 Coordinated patterning mechanisms <strong>of</strong> blood vessels and peripheral nerves<br />
Mukouyama Y.<br />
Stem Cell and Neuro-Vascular Biology, GDBC, NHLBI, NIH<br />
Anatomical proximity and close patterning <strong>of</strong> nerves and blood vessels suggest that there is interdependence<br />
between the two networks. The first such indication <strong>of</strong> this interplay is the responsiveness <strong>of</strong> vascular<br />
development to signals secreted by peripheral sensory nerves in the developing skin. We suggests a<br />
coordinated mechanism in which nerve derived-signals is responsible for vascular patterning signal to recruit<br />
vessels to align with nerves, and for arterial differentiation in the nerve-associated vessels. Interestingly, we<br />
discovered a reciprocal guidance event in the patterning <strong>of</strong> peripheral sympathetic nerves in the developing<br />
heart. Our whole-mount imaging approaches revealed that the pattern <strong>of</strong> large-diameter coronary veins<br />
influences the pattern <strong>of</strong> sympathetic innervation in the developing heart. Further genetic studies and in vitro<br />
organ culture experiments demonstrated that coronary veins serve as an intermediate template that guides<br />
distal sympathetic axon projection via local signal by coronary vascular smooth muscle cells. Our results<br />
suggest that target organs possess unique and stereotypical patterns <strong>of</strong> innervation, mediated by tissue substructures,<br />
such as coronary veins in the heart, that are adapted to complex organ structure and physiology.<br />
4.8 Targeted non-invasive occlusion <strong>of</strong> pharyngeal arch arteries during avian cardiac morphogenesis<br />
Stephanie Lindsey, Huseyin C. Yalcin, Akshay Shekhar, Nozomi Nishimura, Chris B. Schaffer, Jonathan T.<br />
Butcher<br />
Department <strong>of</strong> Biomedical Engineering, Cornell <strong>University</strong><br />
The role <strong>of</strong> hemodynamics in outflow tract and pharyngeal arch artery morphogenesis is poorly understood.<br />
Through the use <strong>of</strong> two- photon microscopy guided femtosecond laser pulses, we nucleated and controlled the<br />
growth <strong>of</strong> microbubbles within outflow vessels without disturbing surrounding tissues. These bubbles<br />
temporarily occluded the vessel, during which time a stable occlusion could be formed by ablating the circulating<br />
thrombocytes that accumulated behind the bubble. These clot-like structures then persisted. Using this<br />
approach, we examine the effects <strong>of</strong> PAA vessel occlusions on embryonic viability, hemodynamic<br />
rearrangement, and downstream outflow tract morphogenesis. We determine that occlusion <strong>of</strong> the right IV<br />
pharyngeal arch artery caused lethality in 99.5% <strong>of</strong> embryos by day 6 (HH28/29), while occlusion <strong>of</strong> the left IV<br />
arch artery was significantly less lethal. In each case, blood flow was redistributed to the III and VI PAA and<br />
vessel diameters were altered significantly. With right IV PAA occlusion, the right III PAA artery increased 47%<br />
in diameter while the right VI decreased 100%. Interestingly, ultrasound derived blood velocity did not change in<br />
the right III PAA but significantly increased in the right VI. No changes in diameter or velocity were seen in the<br />
contralateral PAA. When both IV PAA were occluded, both PAA vessels changed in diameter, resulting in an<br />
overall decrease in ouflow lumen area. These results support that PAA hemodynamics are important<br />
contributor to embryonic development.<br />
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4.9 ORIGIN AND MIGRATION OF ARTERIAL AND VENOUS PROGENITORS FOR THE MAJOR AXIAL<br />
VESSELS<br />
Vikram Kohli 1 , Kira Rehn 1 , Sharina Palencia-Desai 1,2 , and Saulius Sumanas 1,3<br />
1 Division <strong>of</strong> Developmental Biology, Cincinnati Children’s Hospital Medical Center, 2 Molecular and<br />
Developmental Biology Graduate Program, <strong>University</strong> <strong>of</strong> Cincinnati. 3 Division <strong>of</strong> Hematology / Oncology,<br />
Cincinnati Children’s Hospital Medical Center / <strong>University</strong> <strong>of</strong> Cincinnati College <strong>of</strong> Medicine<br />
During the formation <strong>of</strong> the major axial vessels, the dorsal aorta (DA) and the posterior cardinal vein (PCV),<br />
vascular endothelial progenitor cells (angioblasts) adopt an arterial or venous identity prior to circulation.<br />
However, how angioblasts establish their arterial or venous fates and how they migrate to assemble into the<br />
major axial vessels remains controversial. Using the zebrafish as the model organism, we performed single cell<br />
fate mapping and time lapse imaging to investigate the origin <strong>of</strong> angioblasts and their migratory behavior during<br />
the assembly <strong>of</strong> the axial vessels. Our results show that in contrast to the current model, the progeny <strong>of</strong> single<br />
labeled angioblasts can contribute to both the DA and the PCV, and that the arterial or venous identity <strong>of</strong> the<br />
endothelial progenitors is not determined until they migrate towards the midline. We show that angioblasts<br />
originate at different times in two distinct locations, a medial line and a previously unobserved lateral line, both<br />
<strong>of</strong> which exhibit different biases in arterial-venous contribution. Based on cell tracking results, most angioblasts<br />
migrate directly to their arterial and venous positions rather than assembling into a single common precursor<br />
vessel as previously suggested. Using photoactivatable morpholinos, we show that the function <strong>of</strong> the<br />
Etv2/Etsrp transcription factor, a known regulator <strong>of</strong> early vasculogenesis, is required earlier for arterial<br />
differentiation than for venous, which corresponds to the timing <strong>of</strong> the medial and lateral angioblasts migration.<br />
Our results lead to a revised model for arterial-venous differentiation, and will help to dissect the mechanim for<br />
arterial-venous specification.<br />
4.10 Vessel diameter increase during remodeling <strong>of</strong> the mouse yolk sac occurs via vascular fusion and<br />
endothelial cell recruitment<br />
Ryan S. Udan, Tegy J. Vadakkan, and Mary E. Dickinson<br />
Molecular Physiology and Biophysics, Baylor College <strong>of</strong> Medicine<br />
For over the past one hundred years, it has been posited that blood flow regulates the remodeling <strong>of</strong> vessels, and<br />
only recent studies have determined that hemodynamic forces imparted by blood flow are required for vascular<br />
remodeling. However very little is known about the cellular events that promote vessel diameter increase during<br />
remodeling, and how hemodynamic force triggers these events. To assess the cellular events <strong>of</strong> remodeling, we<br />
performed live imaging <strong>of</strong> cultured mouse embryos labeled by two endothelial cell transgenic reporter lines (Flk1-<br />
H2B::eYFP [nucleus] and Flk1-myr::mCherry [membrane]). Our data have revealed that vessels <strong>of</strong> the mouse yolk<br />
sac increase their diameter during remodeling via localized recruitment <strong>of</strong> endothelial cells to growing vessels, and<br />
via vascular fusion, which also acts to refine vessel branches. Hemodynamic force is a critical factor driving vessel<br />
diameter increase as only vessels exposed to high amounts <strong>of</strong> blood flow undergo vessel fusion. Also, arteries<br />
exposed to high blood flow exhibit directional migration <strong>of</strong> endothelial cells upstream <strong>of</strong> the direction <strong>of</strong> blood flow<br />
to hierarchical vessels, and exhibit directional migration <strong>of</strong> endothelial cells towards high flow arteries from lowflow/interconnected<br />
vessels. Whereas, vascular fusion events are absent in embryos exposed to reduced blood<br />
flow (Myosin light chain 2 α mutants), and endothelial cells in these embryos do not exhibit a directional migratory<br />
behavior. Taken together, these results directly show in a mammalian system the cellular changes that occur<br />
during vessel diameter expansion in response to hemodynamic force.<br />
4.11 med23, a subunit <strong>of</strong> transcriptional mediator complex, regulates embryonic angiogenesis and is<br />
essential for embryonic cardiovascular development<br />
Qi Xiao and Zhongzhou Yang<br />
Model Animal Reasearch Center, Nanjing <strong>University</strong>, Nanjing, China<br />
med23, a subunit <strong>of</strong> transcriptional mediator complex, is demonstrated co-regulating expression level <strong>of</strong> various<br />
groups <strong>of</strong> genes in ES and MEF cells with members <strong>of</strong> SPF family. While other candidates binding to med23<br />
remain to be identified. med23 whole body mutant murine embryos was generated to study the role <strong>of</strong> med23 in<br />
embryonic development. Knock-out embryos are lethal at early stage with defects at neural and cardiovascular<br />
system. To further investigate the role <strong>of</strong> med23 in cardiovascular development, we generated med23<br />
conditional knock mice and deleted med23 in endothelial cells specifically with crossing female med23 f/f to<br />
male med23 f/+::Tek-Cre mice. Majority <strong>of</strong> cKO med23 mutant embryos died at two stages. The early group is<br />
lethal between E9.5 and E11. Possible lethal reason is failure formation <strong>of</strong> mature vascular system at placenta<br />
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and yolk sac. The later group is dead between E12.5 and E14 with sever hemorrhage at whole body, especially<br />
at head region, decreased angiogenesis at embryo body and yolk sac and decreased integrity <strong>of</strong> blood vessels.<br />
Normal arterial-venous differentiation was observed in mutant embryos and normal cardiac development was<br />
showed in majority <strong>of</strong> mutant embryos. After contrasting angiogenesis relative genes expression in head region<br />
<strong>of</strong> control and mutant embryos, we detected down-regulation <strong>of</strong> Notch signaling ligand Jag1 and downstream<br />
genes. The phenotype <strong>of</strong> Jag1mutant embryos specific in endothelial is similar with med23, displaying<br />
decreased angiogenesis, dilated small blood vessels, shrink large blood vessels and hemorrhage. We presume<br />
med23 regulates angiogenesis process via regulating Notch pathway. It is reported Jag1 regulates smooth<br />
muscle differentiation as well. We also identified down-regulation <strong>of</strong> αSMA gene expression at yolk sac. To<br />
further investigate details <strong>of</strong> phenotype and mechanism, In vitro experiments <strong>of</strong> endothelial cell assay and in<br />
vivo cell trace experiments will be operated.<br />
4.12 Regulatory mechanisms <strong>of</strong> Semaphorin 3C signaling essential for interaction between cardiac<br />
neural crest and the second heart field<br />
Kazuki Kodo, Keiko Uchida, Takatoshi Tsuchihashi, Hiroyuki Yamagishi<br />
Department <strong>of</strong> Pediatrics, Division <strong>of</strong> Pediatric Cardiology, Keio <strong>University</strong> School <strong>of</strong> Medicine<br />
The cardiac neural crest (CNC) and the second heart field (SHF) play key roles in development <strong>of</strong> the cardiac<br />
outflow tract (OFT), and their interaction to form the outflow tract septum is essential for establishment <strong>of</strong><br />
pulmonary and systemic circulation in higher vertebrates. Recently, we showed that GATA6 directly regulated<br />
the semaphorin 3C (SEMA3C) signaling that is essential for interaction between CNC and SHF during the<br />
development <strong>of</strong> the OFT, and that mutations <strong>of</strong> GATA6 disturbed the activation <strong>of</strong> SEMA3C, resulting in<br />
persistent truncus arteriosus. We further delineated promoter/enhancer <strong>of</strong> Sema3c and identified conserved<br />
regulatory elements for Fox transcription factors within 1kb upstream region and for T-box factors within 2kb<br />
downstream in the 3’UTR, respectively. Interestingly, Foxc1 and Foxc2 activated Sema3c in the OFT region<br />
through direct bindings to their regulatory elements. On the other hand, Tbx1 negatively regulated the<br />
transactivation <strong>of</strong> Sema3c by G ata6. Moreover, the expression <strong>of</strong> Sema3c was ectopically expanded in<br />
pharyngeal mesenchyme in mice with Tbx1 hypomorphic alleles, while it is normally restricted to the SHF in the<br />
pharyngeal mesoderm. These data suggest that Tbx1 may restrict the Sema3c expression in the SHF for<br />
proliferation <strong>of</strong> progenitor cells to form the OFT, and Gata6 and Foxc1/c2 may activate the Sema3c expression<br />
in these cells in a process <strong>of</strong> migration and differentiation into the OFT. Further temporal-spatial molecular<br />
events regulating the Sema3c signaling underlying interaction between CNC and SHF will be discussed.<br />
4.13 SHP2, NEURAL CREST, AND NEONATAL DEATH<br />
Jacquelyn D. Lajiness, Paige Snider, and Simon J. Conway<br />
Medical Scientist Training Program, HB Wells Center for Pediatric Research, Indiana <strong>University</strong> School <strong>of</strong><br />
Medicine, Indianapolis, IN<br />
Src-homology protein tyrosine phosphatase 2 (Shp2) plays diverse roles in many human diseases including<br />
LEOPARD and Noonan syndromes, both <strong>of</strong> which are characterized by neural crest (NC) abnormalities such as<br />
crani<strong>of</strong>acial malformations and congenital heart defects. Shp2 is thought to mediate its functions through a<br />
plethora <strong>of</strong> signaling cascades including Extracellular Regulated Kinases (ERK) 1 and 2. We hypothesize that<br />
abrogation <strong>of</strong> downstream ERK1/2 signaling in NC lineages is primarily responsible for the phenotypes<br />
observed. In order to investigate the role <strong>of</strong> Shp2 and downstream mediators within NC cells, we generated a<br />
unique conditional knockout (cKO) <strong>of</strong> Shp2 in post-migratory NC using a novel Periostin-Cre line engineered in<br />
our lab. Unexpectedly, Shp2 cKOs are indistinguishable from control littermates at birth and exhibit no gross<br />
structural cardiac anomalies. However, around 1-2 weeks after birth, Shp2 cKOs drop <strong>of</strong>f the growth curve and<br />
develop electrocardiogram abnormalities. Significantly, 100% Shp2 cKOs die within 3 weeks after birth. Lineage<br />
mapping using a LacZ reporter mouse (ROSA26r) <strong>of</strong> Cre expression revealed significant hypoplasia <strong>of</strong> the<br />
sympathetic and sensory nervous systems from 3+ days after birth. Specifically, the neural plexus innervating<br />
the heart and the sympathetic chain were reduced. Molecularly, Shp2 cKOs exhibit lineage-specific suppression<br />
<strong>of</strong> activated phospo-ERK1/2 signaling, but not <strong>of</strong> other downstream targets <strong>of</strong> Shp2 such as AKT. These<br />
preliminary data suggest that the hypoplasia <strong>of</strong> the sympathetic and peripheral nervous system, the ECG<br />
abnormalities, and our neonatal death phenotype may all be directly mediated via a loss <strong>of</strong> pERK in postmigratory<br />
NC lineage.<br />
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4.14 Role <strong>of</strong> endocytosis by the neural crest cells in cardiovascular development<br />
Anna Keyte, Harriett Stadt, Kelly Pegram, Margaret L. Kirby, Mary R. Hutson<br />
Duke <strong>University</strong> Department <strong>of</strong> Pediatrics, Division <strong>of</strong> Neonatology<br />
Cardiac neural crest cells (CNCCs) are required for proper remodeling <strong>of</strong> the pharyngeal arch arteries (PAAs),<br />
correct alignment, and septation <strong>of</strong> the cardiac arterial pole. CNCCs are thought to regulate signaling in the<br />
pharynx impacting PAA and secondary heart field (SHF) development. However, it is unknown how this is<br />
accomplished. Signaling pathways involved in cardiovascular development, including Notch, SHH, and FGF,<br />
rely on endocytosis <strong>of</strong> their respective ligands. Here we test the hypothesis that endocytosis by the CNCCs is<br />
required for normal cardiovascular development. An inhibitor <strong>of</strong> clathrin-dependent endocytosis, Pitstop2, was<br />
applied to chick embryos. Embryos displayed double outlet right ventricle (DORV), indicating disrupted<br />
development <strong>of</strong> the SHF, as well as abnormal persistence <strong>of</strong> the fifth and left fourth PAAs, reminiscent <strong>of</strong> human<br />
double aortic arch. Surprisingly, PAA defects occurred most frequently in embryos treated prior to CNCC<br />
migration, long before ve ssel remodeling occurs. Premigratory CNCCs were targeted by electroporation with<br />
either dominant negative Dynamin (dnDyn) or dnRab5a to block endocytosis or vesicle trafficking. Experimental<br />
embryos showed DORV and PAA remodeling defects similar to Pitstop2 treated embryos. Smooth muscle<br />
differentiation within the PAA walls was abnormal. To determine which signaling pathways are impacted by<br />
endocytosis knockdown, RNA was extracted from individual pharynxes for RNA-Seq. This is the first animal<br />
model <strong>of</strong> double aortic arch, and provides a framework for investigating the mechanism by which the CNCCs<br />
remodel the paired PAAs into the asymmetric great arteries. Altogether the evidence indicates a critical role for<br />
endocytosis in proper orchestration <strong>of</strong> cardiovascular development.<br />
4.15 A New Perspective on the Origin and Derivatives <strong>of</strong> Cardiac Neural Crest in Zebrafish<br />
Martha R. Alonzo-Johnsen 1 and Margaret L. Kirby 2<br />
1 Department <strong>of</strong> Biology and 2 Duke <strong>University</strong> Medical Center, Department <strong>of</strong> Pediatrics (Neonatology), Durham,<br />
NC<br />
Cardiac neural crest cells (NCCs) in mouse and chick give rise to the smooth muscle <strong>of</strong> the aortic arch arteries,<br />
form the aorticopulmonary septum and the cardiac ganglia. Zebrafish have unseptated hearts and may not have<br />
cardiac NCCs. We hypothesize that cardiac NCCs contribute to the gill arch arteries and ventral aorta but not to<br />
other heart structures. Two previous studies in zebrafish reported the origin <strong>of</strong> cardiac NCCs and that they gave<br />
rise to myocardium, a fate not observed in chick and mouse. Neither study took a direct approach <strong>of</strong> tracing<br />
known cardiac NCCs. Using the neural crest transgenic Tg(sox10:egfp), we used uncageable rhodamine to<br />
determine the fate <strong>of</strong> gfp-positive NCCs as they migrated from the neural tube. We refined the map <strong>of</strong> the NCCs<br />
that contribute to the branchial arches but found no contribution to the myocardium at 72hpf, as has been<br />
previously reported. We showed that cranial NCCs and cardiac mesoderm were in close proximity (3-5 cell<br />
diameters apart) as NCCs migration begins. Therefore, the previous studies may have spuriously labeled<br />
cardiac mesoderm. Using a Tg(Sox10:Cre) reporter line, which permanently labels cells that express sox10, we<br />
found sox10 positive cells in the heart at 96hpf after all myocardial cells were added. At 120hpf, we found sox10<br />
positive cells in the ventral aorta, similar to NCCs seen in the aortic arch arteries <strong>of</strong> mice and chicks. Cells at<br />
96hpf are likely contributing to innervation <strong>of</strong> the heart, as seen in mouse and chick models.<br />
4.16 Combinatorial Actions <strong>of</strong> Retinoic Acid and Sonic Hedgehog in Cardiac Neural Crest<br />
Song-Chang Lin a , Marie Paschaki b , Pascal Dollé b , Richard Finnell c , and Karen Niederreither c<br />
a Department <strong>of</strong> Cardiology, Baylor College <strong>of</strong> Medicine, Houston; b IGBMC, Illkirch, France; c Dept <strong>of</strong> Nutritional<br />
Sciences, Dell Pediatric Research Institute, The <strong>University</strong> <strong>of</strong> Texas, Austin.<br />
The vitamin A derivative retinoic acid (RA) acts with other embryonic signals to control growth and patterning.<br />
As the RA and Sonic hedgehog (Shh) pathways synergistically control neural progenitor cell survival and<br />
differentiation, we hypothesized similar effects may occur on ectomesenchymal neural crest cells (NCCs). RA<br />
deficiency in Raldh2-/- embryos produces aplasia <strong>of</strong> the 3rd-6th pharyngeal arches. While NCCs form and<br />
initiate their migration, few cells migrate past the defective pharyngeal region towards the heart outflow tract.<br />
Hox genes are regulated by both RA and Shh signaling. Early NCC migration and guidance relies on Hoxa1 and<br />
Hoxb1, whereas genes from paralogy groups 2 to 4 are believed to convey pharyngeal patterning information.<br />
Hoxa1, Hoxa3/d3, Hoxa4/d4, and Hoxb5 levels in the posterior pharyngeal arch region were all reduced under<br />
RA deficiency and these genes were resistant to activation under high level-retinoid treatment. Daily injection <strong>of</strong><br />
the Shh inhi bitor cyclopamine at E7.5-E8.5 effectively blocked the hedgehog targets Ptch1 and Gli1. Combined<br />
Shh inhibition and Raldh2 deficiency reduced the quantity <strong>of</strong> NCCs migrating into the 3th-6th aortic arch region.<br />
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These NCC depletions were also observed in Nkx2.5-Cre-deleted Shh/Raldh2 double mutants and in explant<br />
cultures in which both pathways were blocked. We propose a two-step model in which (i) RA induces multiple<br />
Hox paralogy group expression, and is required for a Hox “code” providing positional identity for caudal<br />
pharyngeal arch patterning; (ii) combined RA and Shh actions are required for ectomesenchymal NCC<br />
progenitors survival, prior to their arrival in the pharyngeal region.<br />
4.17 Roles <strong>of</strong> neural crest-derived fibronectin in cardiovascular development<br />
Xia Wang, Shuan Yu Hou, Sophie Astr<strong>of</strong><br />
Center for Translational Medicine, Jefferson Medicine College<br />
Fibronectin (FN) is essential for vertebrate development, however the function <strong>of</strong> FN in orchestrating<br />
developmental processes is not well understood. Our previous study has shown that cardiac neural crest (CNC)<br />
cell numbers are significantly deficient in FN global null embryos due to defective proliferation and survival <strong>of</strong><br />
CNC progenitors, while migration <strong>of</strong> the CNC is not significantly altered. FN and multipotent neural crest are<br />
unique vertebrate features, and work from our lab showed that FN gene expression is upregulated in the neural<br />
crest before and immediately after neural crest cells exit from the dorsal neural tube. In order to examine the<br />
role <strong>of</strong> FN in neural crest development, we used two different lines <strong>of</strong> mice to ablate FN synthesized by the<br />
neural crest, P3Pro-Cre (neural crest) and AP2a IRES-Cre (neural crest and surface ectoderm). The phenotypes <strong>of</strong><br />
the conditionally mutant embryos were similar when either <strong>of</strong> the above strains was used, indicating that the<br />
observed defects were due to the ablation <strong>of</strong> FN in the neural crest. We found that conditional ablation <strong>of</strong> FN<br />
using either <strong>of</strong> the above strains leads to embryonic lethality and severe cardiovascular abnormalities, including<br />
intra-cardiac cartilage, retroesophageal right subclavian artery, ventricular septum defect, persistent truncus<br />
arteriosus, double outlet right ventricle, and overriding aorta. Although initial migration <strong>of</strong> neural crest cells<br />
occurred normally in the mutant embryos, some FN mutants exhibited decreased numbers <strong>of</strong> CNC cells<br />
compared with their littermate controls. At E11.5, mutant embryos were deficient in neural crest derived smooth<br />
muscle cells around the right fourth aortic arch artery. Further studies will continue to investigate molecular roles<br />
<strong>of</strong> the neural crest cell-synthesized FN in neural crest development and cardiovascular morphogenesis.<br />
4.18 Correlation <strong>of</strong> Great Artery Growth with Hemodynamic Wall Shear Rate in the Developing Mouse<br />
Fetus: a Study using Ultrasound Imaging, EFIC Reconstructions and Computational Modeling.<br />
Choon Hwai Yap 1 , Xiao Qin Liu 1 , Rod Bunn 2 , Kerem Pekkan 3 , Cecilia Lo 1<br />
1 Department <strong>of</strong> Developmental Biology, <strong>University</strong> <strong>of</strong> Pittsburgh School <strong>of</strong> Medicine, Pittsburgh. 2 Vashaw<br />
Scientific Inc., Norcross, GA. 3 Department <strong>of</strong> Biomedical Engineering, Carnegie Mellon <strong>University</strong>, Pittsburgh<br />
Introduction: Adverse fluid mechanical environment <strong>of</strong> the fetal cardiovascular system has been hypothesized<br />
to play a role in structural heart malformations. We present a study <strong>of</strong> the geometry and fluid mechanics <strong>of</strong> fetal<br />
great arteries during normal fetal development from embryonic day 14.5 (E14.5) to near term. Methods:<br />
Ultrasound bio-microscopy (UBM) was used to measure blood velocity <strong>of</strong> the great arteries. Subsequently,<br />
specimens were cryo-embedded and sectioned using episcopic fluorescent image capture (EFIC) to obtain<br />
high-resolution 2D serial image stacks, which were used for 3D reconstructions and quantitative measurement<br />
<strong>of</strong> great artery and aortic arch dimensions. EFIC and UBM data were input into computational fluid dynamics<br />
(CFD) for modeling hemodynamics. Results: In normal mouse fetuses between E14.5-18.5, ultrasound<br />
imaging showed gradual increase in blood velocity in the aorta, pulmonary trunk, and descending aorta.<br />
Measurement by EFIC imaging showed a similar gradual increase in cross sectional area <strong>of</strong> these vessels. CFD<br />
modeling showed great artery hemodynamic wall shear rate to be 2900-3300 sec -1 at peak flow. Average wall<br />
shear rate remained at this level despite vessel growth over these stages. Conclusion: We observed the<br />
maintenance <strong>of</strong> a constant wall shear rate during mid to late mouse fetal development. These results suggest a<br />
homeostatic sensing mechanism that may link hemodynamic shear to the regulation <strong>of</strong> vessel growth. Further,<br />
insights into fetal cardiovascular fluid mechanics may help elucidate whether fetal cardiovascular surgery may<br />
have benefit for patients with congenital heart disease. (Study supported by U01-HL098180)<br />
4.19 The role <strong>of</strong> Forkhead Box transcription factors Foxc1 and Foxc2 in myocardial wound repair<br />
Kyoo Seok Shim, Hyun-Young Koo, Alex Hanna, Ting Liu, Aiko Ito, Carey Nassano-Miller, Sol Misener,<br />
Christine Kamide, Douglas Losordo and Tsutomu Kume<br />
Feinberg Cardiovascular Research Institute, Feinberg School <strong>of</strong> Medicine, Northwestern <strong>University</strong>, <strong>Chicago</strong>, IL<br />
Summary: Forkhead Box (Fox) transcription factors are essential for regulating cell growth, proliferation,<br />
differentiation, and longevity. Among the large subclasses (FoxA to FoxS) <strong>of</strong> Fox transcription factors, Foxc1<br />
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and Foxc2 are known as important regulators for cardiovascular development. However, the role <strong>of</strong> Foxc1 and<br />
Foxc2 in pathological neovascularization involving ischemic condition remains still unknown. Here, we<br />
employed a mouse myocardial infarction (MI) model to study the role <strong>of</strong> Foxc1 and Foxc2 in cardiac tissue<br />
repair following ischemic insult. Methods: Foxc1 and Foxc2 heterozygous mutant (+/-) and wild-type mice were<br />
used for this study, and MI was induced surgically by ligating the left anterior descending coronary artery. To<br />
examine heart functions, we measured fractional shortening and ejection fraction at day 7, 14 and 28 after MI<br />
using echocardiography. After 4 weeks post-surgery, histological sections <strong>of</strong> heart samples were obtained to<br />
observe fibrosis with Masson trichrome staining. Results: There was no significant difference in infarction areas<br />
between Foxc1+/- and wild-type mice, and the ejection fraction and fractional shortening did not differ between<br />
the two groups at each time point. In contrast, echocardiographic measurements <strong>of</strong> left ventricular ejection<br />
fractions and fractional shortening were reduced in Foxc2+/- mice after MI compared to wild-type mice, whereas<br />
cardiac fibrosis was increased in Foxc2+/- mice. Most significantly, Foxc2+/- mice had a drastic reduction in<br />
capillary vessel formation in the ischemic border zone after MI compared to wild-type mice. Conclusions: Our<br />
results indicate that Foxc2 is more involved in neovascularization during ischemic tissue repair after MI than<br />
Foxc1.<br />
4.20 Intrinsic non-mesothelial origin <strong>of</strong> endothelial cells in the avian intestine.<br />
Nichelle Winters 1 , David Bader 1,2<br />
1 Department <strong>of</strong> Cell and Developmental Biology, Vanderbilt <strong>University</strong><br />
2 Department <strong>of</strong> Medicine, Vanderbilt <strong>University</strong><br />
The proepicardium <strong>of</strong> the heart contributes at least a subpopulation <strong>of</strong> coronary endothelial cells to the<br />
developing heart. The avian intestine does not derive its mesothelial lining from a proepicardial-like structure.<br />
Instead, mesothelial progenitors are resident and broadly distributed throughout the intestinal primordium. We<br />
investigated the mechanisms <strong>of</strong> intestinal vascular formation to determine if similar variation could be observed<br />
between the heart and intestine in endothelial origins. We labeled intestinal mesothelial progenitors with a<br />
replication incompetent retrovirus and allowed the embryos to develop for up to two weeks. While we observed<br />
vascular smooth muscle cells and pericytes among the progeny <strong>of</strong> labeled mesothelial progenitors, we did not<br />
identify any endothelial descendants. We also transplanted quail splanchnopleure prior to mesothelial formation<br />
into the coelomic cavity <strong>of</strong> chick embryos. The grafted splanchnopleure generated a gut tube complete with a<br />
mesentery and vasculature. We determined that endothelial cells <strong>of</strong> the graft were derived primarily from the<br />
graft and not from the host. Injections <strong>of</strong> lectin and a vital dye directly into the heart <strong>of</strong> HH14-17 quail embryos<br />
also demonstrated the endothelial plexus <strong>of</strong> the splanchnopleure is in communication with the systemic<br />
vasculature. Furthermore, intracardiac injection <strong>of</strong> a replication incompetent retrovirus at HH14-17 resulted in<br />
labeled endothelial cells throughout the vascular tree <strong>of</strong> the intestine demonstrating endothelial cells <strong>of</strong> the early<br />
splanchnopleure are remodeled to generate the mature vasculature. Thus, we conclude the majority <strong>of</strong> avian<br />
intestinal endothelial cells are derived from remodeling <strong>of</strong> the primitive endothelial plexus <strong>of</strong> the<br />
splanchnopleure.<br />
4.21 A comprehensive timeline <strong>of</strong> endothelial development in the small intestine <strong>of</strong> the quail embryo<br />
Rebecca Thomason, Niki Winters, David Bader<br />
Department <strong>of</strong> Medicine, Vanderbilt <strong>University</strong><br />
The early embryonic gut is a tube composed <strong>of</strong> splanchnic mesoderm and endoderm. Many studies have<br />
investigated specific tissue layers or time points in gut development. However, an inclusive timeline <strong>of</strong><br />
endothelial cell development and the simultaneous events that occur throughout small intestine development is<br />
lacking. We utilized immun<strong>of</strong>luorescence, morphometric analysis, histology, and transgenic quail embryos to<br />
examine the relationship <strong>of</strong> endothelial plexus formation to mesothelial formation, mesenchymal growth, and<br />
smooth muscle morphogenesis. Using Tg(tie1:H2B-eYFP) quail embryos, the nuclei <strong>of</strong> endothelial cells are first<br />
visualized in the mesenchymal space at E2.1, between two basement membranes, lining the endoderm and<br />
splanchnic mesoderm, in the open gut tube. At E6, two concentric rings <strong>of</strong> endothelial cells are observed, at the<br />
same time point the gut tube closes, and when both a mesothelium and visceral smooth muscle are first<br />
observed. At E11, the major blood vessels, circumferentially covering the midgut, are first detected, and smooth<br />
muscle cells develop around the YFP+ cells, indicating muscularization <strong>of</strong> the vessels. We observed endothelial<br />
cells in the villi at E14, at which smooth muscle actin staining is also visualized for the first time in the villi. Taken<br />
together, these data provide a comprehensive timeline <strong>of</strong> endothelial development in the small intestine.<br />
Normally, developmental processes not correlated to one another may have both a temporal and morphological<br />
117
elationship. This timeline will benefit both researchers examining intestinal development and clinicians studying<br />
congenital syndromes that may originate from a combination <strong>of</strong> disrupted developmental processes.<br />
4.22 SEMAPHORIN3D PATTERNS THE PULMONARY VEINS<br />
Karl Degenhardt 1 , Manvendra K. Singh 2 , Haig Aghajanian 2 , Daniele Massera 2 , Qiaohong Wang 2 , Jun Li 2 , Li Li 2 ,<br />
Amanda Phillips 2 , Lauren J. Francey 3 , Ian D. Krantz 3 , Peter J. Gruber 4 and Jonathan A. Epstein 2<br />
1 Divisions <strong>of</strong> Cardiology and 3 Human Genetics, Department <strong>of</strong> Pediatrics, Children’s Hospital <strong>of</strong> Philadelphia,<br />
2 Department <strong>of</strong> Cell and Developmental Biology, Cardiovascular Institute and Institute for Regenerative<br />
Medicine, Perelman School <strong>of</strong> Medicine at the <strong>University</strong> <strong>of</strong> Pennsylvania, 1154 BRB II, 421 Curie Blvd.<br />
Philadelphia, PA 19104 USA, and 4 Departments <strong>of</strong> Surgery and Molecular Medicine, <strong>University</strong> <strong>of</strong> Utah School<br />
<strong>of</strong> Medicine, Salt Lake City, UT<br />
A fundamental aspect <strong>of</strong> mammalian and avian cardiopulmonary physiology is the complete separation <strong>of</strong><br />
systemic and pulmonary circulations. Establishment <strong>of</strong> this highly efficient oxygen delivery system requires both<br />
intracardiac septation and proper vascular patterning during embryogenesis. Total anomalous pulmonary<br />
venous connection (TAPVC) is a potentially lethal congenital heart disease that occurs when the pulmonary<br />
veins fail to connect normally to the left atrium, allowing mixing <strong>of</strong> pulmonary and systemic blood. In contrast to<br />
the broad knowledge <strong>of</strong> arterial vascular patterning, little is known about the patterning <strong>of</strong> veins. Here we show<br />
that the secreted guidance molecule Semaphorin 3d (Sema3d) is crucial for normal patterning <strong>of</strong> the pulmonary<br />
veins. Prevailing models suggest that TAPVC occurs when the midpharyngeal endothelial strand, the precursor<br />
<strong>of</strong> the common pulmonary vein, fails to form at the proper location on the ventral surface <strong>of</strong> the embryonic<br />
common atrium. However, analysis <strong>of</strong> Sema3D mutant embryos shows that TAPVC occurs despite normal<br />
formation <strong>of</strong> the midpharyngeal endothelial strand. Rather, the emerging venous plexus destined to form the<br />
pulmonary veins fails to anastomose uniquely with the properly formed midpharyngeal endothelial strand and<br />
forming endothelial tubes penetrate a boundary normally produced by Sema3d expression, resulting in aberrant<br />
connections. These results identify Sema3d as a critical pulmonary venous patterning cue and provide<br />
experimental evidence for an alternate developmental model to explain abnormal pulmonary venous<br />
connections.<br />
4.23 A mouse fetus with a double lumen aortic arch malformation<br />
Stefan H. Geyer, Barbara Maurer, Birgit Zendron, Wolfgang J. Weninger<br />
Center for Anatomy and Cell Biology, Medical <strong>University</strong> <strong>of</strong> Vienna<br />
Double lumen aortic arches are rarely diagnosed in humans. They are considered to result from the abnormal<br />
persistence <strong>of</strong> a left 5 th pharyngeal arch artery (PAA). Although the mouse is an important model for researching<br />
the genesis <strong>of</strong> PAA malformations, there is no documented case <strong>of</strong> a persisting 5 th PAA in mice. This study aims<br />
at presenting a mouse fetus <strong>of</strong> the him:OF1 strain, which developed a double lumen aortic arch. In order to<br />
comprehensively characterize the malformation, we created surface rendered three-dimensional (3D) computer<br />
models <strong>of</strong> the great intrathoracic arteries with the aid <strong>of</strong> the “High-resolution episcopic microscopy” (HREM)<br />
technique. The voxel size <strong>of</strong> the 3D models was 1.07 x 1.07 x 2µm 3 . The gross anatomy and the dimensions <strong>of</strong><br />
the great intrathoracic arteries <strong>of</strong> the malformed fetus appeared to be normal with one exception. The segment<br />
<strong>of</strong> the aortic arch between the origin <strong>of</strong> the left common carotid artery and the left subclavian artery consisted <strong>of</strong><br />
two channels. The length <strong>of</strong> the segment and the sum <strong>of</strong> the cross sectional areas <strong>of</strong> the two channels were<br />
approximately the same as the length and the cross sectional area <strong>of</strong> the corresponding one channeled aortic<br />
arch segment <strong>of</strong> normal fetuses. Our results show that mice, like humans, do show double lumen aortic arch<br />
malformations. They also show that this malformation does not affect the formation and remodeling <strong>of</strong> the<br />
arteries located proximally and distally.<br />
4.24 Mouse embryos feature a 5 th pair <strong>of</strong> pharyngeal arch arteries<br />
Wolfgang J. Weninger, Michael Dorn, Barbara Maurer, Stefan H. Geyer<br />
Center for Anatomy and Cell Biology, Medical <strong>University</strong> <strong>of</strong> Vienna<br />
Recent studies do suggest that mouse embryos do not develop a 5 th pair <strong>of</strong> pharyngeal arch arteries (PAAs). If<br />
true, the suitability <strong>of</strong> the mouse as a model for researching malformations <strong>of</strong> the great intrathoracic arteries<br />
would be degraded. The aim <strong>of</strong> our study was to evaluate whether mouse embryos do or do not develop a 5 th<br />
pair <strong>of</strong> PAAs. We analysed the vascular phenotype <strong>of</strong> 30 mouse embryos aged 12-12.5 days post conception<br />
(dpc) with the aid <strong>of</strong> volume data and three-dimensional (3D) computer models generated with the “High<br />
resolution episcopic microscopy” technique. The embryos were <strong>of</strong> the Him:OF1 strain. The digital volume data<br />
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had a voxel size <strong>of</strong> 1.07x1.07x2 µm 3 . In half <strong>of</strong> the embryos (15 <strong>of</strong> the 30) we detected a thick vascular channel,<br />
which connected the lumen <strong>of</strong> the 4 th and the lumen <strong>of</strong> the 6 th pharyngeal arch arteries. Nine embryos (30%)<br />
showed this channel unilaterally. Six embryos (20%) showed it bilaterally. According to descriptions <strong>of</strong> the<br />
human 5 th pharyngeal arch artery and to descriptions <strong>of</strong> the 5 th pharyngeal arch artery in rat embryos, we<br />
identified this vascular channel as the 5 th pharyngeal arch artery. Our study demonstrates that mice do develop<br />
a <strong>of</strong> 5 th pair <strong>of</strong> PAAs around 12.5 dpc. Hence it re-establishes the reputation <strong>of</strong> the mouse as an excellent model<br />
organism for researching the genesis <strong>of</strong> malformations <strong>of</strong> the great intrathoracic arteries <strong>of</strong> humans.<br />
4.25 The phenotype <strong>of</strong> the pharyngeal arch arteries defines a new type <strong>of</strong> cephalothoracopagus<br />
embryos<br />
B. Maurer, S.H. Geyer, K. Dorfmeister, and W.J. Weninger<br />
Center for Anatomy and Cell Biology, Integrative Morphology, Medical <strong>University</strong> <strong>of</strong> Vienna, Vienna, Austria<br />
The presentation aims at providing a gross description <strong>of</strong> the phenotype <strong>of</strong> a cephalothoracopagus chick<br />
embryo <strong>of</strong> developmental stage 31 according to Hamburger Hamilton (HH) and a detailed metric analysis <strong>of</strong> its<br />
great intrathoracic arteries. It further aims at providing reference data, which define the normal dimensions <strong>of</strong><br />
the great arteries at HH31. We used the high-resolution episcopic microscopy (HREM) technique for creating<br />
virtual three-dimensional (3D) models <strong>of</strong> 5 normally developed and one spontaneously developed<br />
cephalothoracopagus chick embryo. The models were employed for topologic analysis <strong>of</strong> the organs and blood<br />
vessels and for measuring the diameters <strong>of</strong> the lumina <strong>of</strong> the great intrathoracic arteries. The measurements<br />
were performed following a recently proposed measuring protocol. The cephalothoracopagus embryo showed<br />
two notochords, two spinal chords and two dorsal aortae. It had a single, four-chambered heart, a single<br />
respiratory tract, and a single upper alimentary tract. The topology <strong>of</strong> the pharyngeal arch arteries was normal.<br />
The dimensions <strong>of</strong> the lumina <strong>of</strong> the great intrathoracic arteries were similar to that <strong>of</strong> the control embryos. The<br />
phenotype <strong>of</strong> the cephalothoracopagus embryo does not match classical descriptions and cannot be entirely<br />
explained with any <strong>of</strong> the accepted theories. The embryo thus must be considered as having a yet unknown<br />
type <strong>of</strong> cephalothoracopagus malformation.<br />
4.26 Molecular and Functional Pr<strong>of</strong>iles <strong>of</strong> VSMCs Based on Embryonic Origin<br />
Elise R. Pfaltzgraff 1 , J. Jeff Reese 2 , David M. Bader 1,3 , and Patricia A. Labosky 1<br />
1 Center for Stem Cell Biology, Department <strong>of</strong> Cell and Developmental Biology, 2 Department <strong>of</strong> Pediatrics,<br />
3 Department <strong>of</strong> Medicine, Vanderbilt <strong>University</strong> Medical Center, Nashville, TN<br />
Vascular smooth muscle cells (VSMCs) in the aorta are derived from two embryonic origins: the cardiac neural<br />
crest (CNC) and the mesoderm. The CNC contributes VSMCs to the ascending aorta and aortic arch (aAo)<br />
while the mesoderm contributes VSMCs to the descending aorta (dAo) distal to the ductus arteriosus. These<br />
two populations do not mix and remain compartmentalized through adulthood. Based on our preliminary and<br />
published data demonstrating distinct differences in VSMC gene expression and contractility, our hypothesis is<br />
that NC-derived VSMCs have molecular and functional characteristics distinct from mesoderm-derived VSMCs.<br />
Using myographic approaches, we established fundamental physiological differences between these two<br />
vessels in the embryo; the dAo is more contractile than the aAo. We conducted a microarray comparing these<br />
two regions <strong>of</strong> the embryonic aorta, and the results support our central hypothesis that aAo and dAo have<br />
distinct gene expression pr<strong>of</strong>iles. We have verified the expression <strong>of</strong> many <strong>of</strong> these genes with qRT-PCR. To<br />
confirm the functional difference between NC- and mesoderm-derived VSMCs, we have generated clonal VSMC<br />
lines from each region <strong>of</strong> the aorta and are assaying their contractile and migratory characteristics in vitro.<br />
Together these experiments will lead to discovery <strong>of</strong> molecular criteria to classify VSMCs and establish<br />
functional differences between VSMCs <strong>of</strong> distinct embryonic origins.<br />
4.27 Tenascin-C may protect vascular wall as a molecular shock-absorber.<br />
Kyoko Imanaka-Yoshida 1)2) , Taizo Kimura 3) , Koichi Yoshimura 3)4) , Toshimichi Yoshida 1)2) ,Hiroki Aoki 3)5)<br />
1) Mie <strong>University</strong> Research Center for Matrix Biology, 2) Department <strong>of</strong> Pathology and Matrix Biology, Mie<br />
<strong>University</strong> Graduate School <strong>of</strong> Medicine, Tsu, Japan. 3) Department <strong>of</strong> Molecular Cardiovascular Biology and<br />
4) Cardiovascular Division <strong>of</strong> Cardiology, Department <strong>of</strong> Medicine and Clinical Science, Yamaguchi, Ube,<br />
Japan 5) , <strong>University</strong> Graduate School <strong>of</strong> Medicine Research Institute, Kurume <strong>University</strong>, Kurume, Japan<br />
Tenascin-C (TnC)is an extracellular glycoprotein, generally expressed at high levels during embryonic<br />
development in a spatiotemporal-restricted manner, sparsely detected in normal adult, but re-expressed in<br />
pathological condition in various tissue playing significant roles in tissue remodeling. During vascular<br />
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development, TnC is highly upregulated associated with smooth muscle/mural cell recruitment, however, the<br />
expression pattern is complex, <strong>of</strong>ten showing high sensitivity to mechanical stress. In normal adult mice, TnC is<br />
constitutively expressed in the abdominal aorta, but not in the proximal aorta. Biomechanical analyses revealed<br />
the significant reduction <strong>of</strong> flexibility <strong>of</strong> abdominal aorta <strong>of</strong> TnC knock out (TnC KO) mice to compare with that <strong>of</strong><br />
the wild type, although TnC knock mice do not show distinct phenotype in the normal state. We created a<br />
mouse model <strong>of</strong> aortic stiffening by a combination <strong>of</strong> periaortic application <strong>of</strong> calcium chloride in the infrarenal<br />
aorta and systemic administration <strong>of</strong> angiotensin II (Ca+AngII). Ca+AngII treatment upregulated TnC expression<br />
in the upper aorta, and eventually induced marginal dilataion <strong>of</strong> the suprarenal aorta. In TnC KO, Ca+AngII<br />
treatment caused striking enlargement <strong>of</strong> the suprarenal aorta. Histological section <strong>of</strong> the enlarged suprarenal<br />
aorta showed a large medial dissection with "double barrel" appearance, while the wildtype showed only modest<br />
medial thickening and adventitial fibrosis. Transcriptome analysis indicated the reduced matrix deposition and<br />
exaggerated proinflammatory response in suprarenal TnC KO aorta before dissection. These results suggest<br />
TnC may work as a stress-activated molecular damper to keep the destructive stress response <strong>of</strong> vascular wall.<br />
4.28 Valentine-like is a novel component <strong>of</strong> the CCM pathway and is essential for cardiovascular<br />
development in zebrafish.<br />
Jonathan Rosen, Vanessa Sogah, Li Ye, John Mably<br />
Children's Hospital Boston, Harvard Medical School<br />
The cerebral cavernous malformation (CCM) pathway is required for normal cardiovascular development in<br />
zebrafish and mouse. We have previously shown that zebrafish embryos mutant for the CCM pathway<br />
genes heart <strong>of</strong> glass (heg), santa (san), or valentine (vtn) exhibit a massive dilation <strong>of</strong> the heart chambers and<br />
inflow tract and lack blood circulation. Recently, we have identified a novel, conserved gene bearing<br />
considerable sequence similarity to vtn, which we have named valentine-like (vtnl). Morpholino knockdown<br />
<strong>of</strong> vtnl confers a phenotype resembling that <strong>of</strong> the CCM pathway mutants; affected embryos have<br />
moderately enlarged atria and inflow tracts and lack circulation. Co-injection <strong>of</strong> vtnl and san morpholinos at<br />
concentrations that individually cause no phenotype results in characteristic cardiovascular defects,<br />
defining vtnl as an enhancer <strong>of</strong> the CCM phenotype. Overexpression <strong>of</strong> vtn can partially rescue vtnl morphant<br />
defects, suggesting that the two genes serve similar functions in vivo. vtn and vtnl both encode proteins<br />
containing phospho-tyrosine binding (PTB) domains, and we have shown in cell culture that Vtnl binds San in an<br />
interaction requiring the N-terminal region <strong>of</strong> San, which contains two NPxY motifs that are likely PTB targets.<br />
Mutations in the human homologs <strong>of</strong> san (CCM1), vtn (CCM2), and one other gene (CCM3) have previously<br />
been shown to cause cerebral cavernous malformations, a relatively common vascular anomaly. However,<br />
many CCM patients have no mutations in these genes. We propose that the human homolog <strong>of</strong> vtnl,<br />
C20ORF160, is an excellent candidate to be investigated for causative mutations in patients with cerebral<br />
cavernous malformations.<br />
4.29 Hemogenic activity <strong>of</strong> the endocardium is regulated by Nkx2-5<br />
Haruko Nakano 1,2,3,4 , Armin Arshi 1 , Jae-Ho Shin 1 , Xiaoqian Liu 1 , Ben van Handel 1 , Rajkumar Sasidharan 1 ,<br />
Andrew W. Harmon 1,4 , Yasuhiro Nakashima 1 , Robert J. Schwartz 5 , Simon J. Conway 6,7 , Richard P. Harvey 8,9 ,<br />
Mohammad Pashmforoush 10 , Hanna K. A. Mikkola 1,2,3,4 , Atsushi Nakano 1,2,3,4<br />
1 Department <strong>of</strong> Molecular Cell and Developmental Biology, <strong>University</strong> <strong>of</strong> California, Los Angeles, Los Angeles, CA 90095,<br />
USA. 2 Eli and Edythe Broad Center <strong>of</strong> Regenerative Medicine and Stem Cell Research, <strong>University</strong> <strong>of</strong> California, Los<br />
Angeles, Los Angeles, CA 90095, USA. 3 Jonsson Comprehensive Cancer Center, <strong>University</strong> <strong>of</strong> California, Los Angeles, Los<br />
Angeles, CA 90095, USA. 4 Molecular Biology Institute, <strong>University</strong> <strong>of</strong> California, Los Angeles, Los Angeles, CA 90095, USA.<br />
5 Department <strong>of</strong> Biology and Biochemistry, <strong>University</strong> <strong>of</strong> Houston, Houston, 77204, USA. 6 Departments <strong>of</strong> Anatomy & Cell<br />
Biology, Medical & Molecular Genetics, Biochemistry & Molecular Biology, Indianapolis, IN 46202, USA. 7 Herman B Wells<br />
Center for Pediatric Research, Indiana <strong>University</strong> School <strong>of</strong> Medicine, Indianapolis, IN 46202, USA. 8 Developmental Biology<br />
Division, the Victor Chang Cardiac Research Institute, Darlinghurst, New South Wales, 2010, Australia. 9 St Vincent's Clinical<br />
School, <strong>University</strong> <strong>of</strong> New South Wales, Australia. 10 Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell<br />
Research, <strong>University</strong> <strong>of</strong> Southern California Keck School <strong>of</strong> Medicine, 90089<br />
We have previously demonstrated that single cardiac progenitors expressing Flk1 Isl1, and Nkx2.5 give rise to<br />
both cardiac and endocardial/endothelial cell lineages during early murine cardiogenesis. Interestingly, the pool<br />
<strong>of</strong> Flk1+/Isl1+/Nkx2.5+ cardiac progenitors express multiple hematopoietic transcription factors. Interestingly, the<br />
pool <strong>of</strong> Flk1+/Isl1+/Nkx2.5+ cardiac progenitors express multiple hematopoietic transcription factors. Although a<br />
close relationship between cardiac, endocardial, and hematopoietic lineages has been suggested in Drosophila,<br />
zebrafish, and embryonic stem cell in vitro differentiation models, the activity <strong>of</strong> hematopoietic genes expressed<br />
in the developing mammalian heart remains unclear. Here, we examined the hemogenic activity <strong>of</strong> the<br />
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developing heart. Mouse heart explants from pre-circulation stages and Ncx1 mutant embryos generated<br />
myeloid and erythroid colonies in the absence <strong>of</strong> circulation. The hemogenic activity arose from a subset <strong>of</strong><br />
endocardial cells in the outflow tract and atria earlier than in the aorta-gonad-mesonephros region. Furthermore,<br />
we found that a cardiac homeobox transcription factor, Nkx2-5, was expressed in and required for a hemogenic<br />
subset <strong>of</strong> the endocardium and yolk sac endothelium during embryogenesis. Together, these data suggest that<br />
Nkx2-5-dependent subset <strong>of</strong> endocardial and yolk sac endothelial cells serve as a de novo source for<br />
hematopoietic progenitors.<br />
4.30 Characterization <strong>of</strong> the cardiac lymphatic system<br />
Klotz, Linda<br />
<strong>University</strong> College London<br />
The lymphatic vasculature is a blind-ended network that is crucial for tissue fluid homeostasis, immune<br />
surveillance and lipid adsorption from the gut. The heart is the first, and conceivably most important, organ to<br />
develop in humans. However, the mechanisms regulating the development <strong>of</strong> the cardiac lymphatic vessels, as<br />
well as their cellular origin, are yet to be described. In this study, we shed light on the development and origin <strong>of</strong><br />
the cardiac lymphatics from mid-gestation to early adulthood by utilizing immunohistochemistry and optical<br />
projection tomography (OPT). We show that lymphatic precursors are present from E14.5 in the epicardium, and<br />
vessels first sprout at E14.5 in the outflow region <strong>of</strong> the heart. Cardiac lymphatic vessels closely follow the path<br />
<strong>of</strong> coronary vessels as they develop, and by P15 the lymphatic vasculature encompasses the entire heart.<br />
Lineage tracing analysis suggests a non-endothelial origin for developmental de novo lymphvasculogenesis in<br />
the heart and indicates a putative involvement <strong>of</strong> the myeloid lineage. Our characterization <strong>of</strong> the cardiac<br />
lymphatics <strong>of</strong>fers a novel insight into a largely overlooked, but arguably very important part <strong>of</strong> the lymphatic<br />
system. Future studies will focus on applying the findings from lymphatic development in the embryonic heart to<br />
adult cardiovascular pathology and disease.<br />
4.31 Endothelin-1/Endothelin type-A receptor signaling regulates pharyngeal arch artery development<br />
through Dlx5/6-independent pathway.<br />
Ki-Sung Kim 1,2 , Koichi Nishiyama 1 , Yuichiro Arima 1 , Rieko Asai 1 , Yasunobu Uchijima 1 , Yukiko Kurihara 1 ,<br />
Takahiro Sato 1 , Takashi Igarashi 2 and Hiroki Kurihara 1 .<br />
1 Departmet <strong>of</strong> Physiological Chemistry and Metabolism, Graduate School <strong>of</strong> Medicine, The <strong>University</strong> <strong>of</strong> Tokyo.<br />
2 Department <strong>of</strong> Pediatrics, Graduate School <strong>of</strong> Medicine, The <strong>University</strong> <strong>of</strong> Tokyo<br />
Endothelin-1 (Edn1) is a vasoconstrictor peptide, and is also involved in the development <strong>of</strong> cranial/ cardiac<br />
neural crest-derived tissues and organs. In crani<strong>of</strong>acial development, Edn1 binds to Endothelin type-A receptor<br />
(Ednra) to induce homeobox genes Dlx5/Dlx6 and determines the mandibular identity in the first pharyngeal<br />
arch. However, it remains unsolved whether this pathway is also involved in pharyngeal arch artery<br />
development to form the thoracic arteries. Here, we show that the abnormal branches from the common carotid<br />
arteries were observed in Edn1 or Ednra knock-out embryos, but not in Dlx5/Dlx6 double knock-out embryos.<br />
These anomalies derived from abnormal persistence <strong>of</strong> the first and second pharyngeal arch arteries. Abnormal<br />
smooth muscle cell differentiation from the neural crest cells was observed at the first and second pharyngeal<br />
arch arteries <strong>of</strong> Ednra knock-out embryos. These findings indicate that the formation <strong>of</strong> pharyngeal arch arteries<br />
does not depend o n Dlx5/Dlx6-mediated ventral identification <strong>of</strong> the pharyngeal arch, and the ET1/Ednra signal<br />
regulates the properly-directed differentiation <strong>of</strong> the neural crest cells into smooth muscle cells in the pharyngeal<br />
arch arteries.<br />
4.32 Protection <strong>of</strong> oral hydrogen water as an antioxidant on pulmonary hypertension<br />
Bin He, 1* Yufeng Zhang, 2* Bo Kang, 2 Jian Xiao, 2 Bing Xie, 3 and Zhinong Wang 2<br />
1 Department <strong>of</strong> Anesthesiology and SICU, Xinhua Hospital, Shanghai Jiaotong <strong>University</strong> School <strong>of</strong> Medicine,<br />
Shanghai 200092, China. 2 Department <strong>of</strong> Cardiothoracic Surgery, Changzheng Hospital, Second Military<br />
Medical <strong>University</strong>, Shanghai 200003, China. 3 Department <strong>of</strong> Burn, Changhai Hospital, the Second Millitary<br />
Medical <strong>University</strong>, Shanghai 200433, China<br />
Background: This study was to explore protection <strong>of</strong> hydrogen as an antioxidant on monocrotaline (MCT)induced<br />
pulmonary hypertension (PH). Instead <strong>of</strong> hydrogen-rich saline, we made new modification by using oral<br />
hydrogen water (HW), which is more convenient, less costly and longer time to be released. Methods: Thirty-six<br />
SD rats were equally randomized to three groups: SHAM group, MCT group and MCT+HW group. The PH<br />
model was established by intraperitoneal injection <strong>of</strong> MCT and the mean pulmonary arterial pressure (mPAP)<br />
121
was measured two weeks later. MCT group were administered with ordinary drinking water, and MCT+HW<br />
group were administered with HW. Another two weeks later, the ventricular weight, left ventricular+septum<br />
weight, and right ventricle weight (RV) were measured. The right ventricular hypertrophy index (RVHI) was<br />
calculated. Damage to the lung tissue was observed and scored by HE staining. Changes <strong>of</strong> 3-nitrityrosine (3nt)<br />
and Intercellular Adhesion Molecule-1 (ICAM -1) in the lung tissue were observed by immunohistochemistry.<br />
Results: mPAP, RV and RVHI in MCT group were significant higher than those in SHAM group (P < 0.05).<br />
mPAP, while RV and RVHI in MCT+HW group were significantly lower than those in MCT group (P < 0.05). In<br />
addition, 3-nt and ICAM-1 were increased significantly in MCT group and decreased significantly in MCT+HW<br />
group (P < 0.05). Conclusions: Oral hydrogen water has a significant protective effect on MCT-induced PH,<br />
which is more convenient to use than hydrogen-rich saline. And this effect is associated with the antioxidative<br />
ability <strong>of</strong> hydrogen and its action <strong>of</strong> reducing pulmonary inflammatory response.<br />
4.33 Identification <strong>of</strong> a minimal arterial enhancer <strong>of</strong> Dll4 and implications for artery-vein specification.<br />
Joshua D. Wythe, William Schachterle, W. Patrick Devine, John N. Wylie, Didier Y. Stainier, Brian L. Black,<br />
Benoit G. Bruneau<br />
Gladstone Institute <strong>of</strong> Cardiovascular Disease<br />
The transcriptional regulation <strong>of</strong> arteriovenous specification is not known. An essential role for VEGF and Notch<br />
signaling in establishing and maintaining vessel identity has been well-established. VEGF induces arteryspecific<br />
expression <strong>of</strong> Dll4 and activates Notch signaling to promote arteriogenesis. In the mouse, Dll4 is the first<br />
arterial marker, and is required for arteriovenous specification. The transcriptional mediators that induce Dll4<br />
expression remain unclear. To elucidate the transcriptional basis <strong>of</strong> artery-specific gene expression, we have<br />
employed transgenic reporter assays to identify the regulatory elements that drive artery-specific expression <strong>of</strong><br />
Dll4 in mouse development. The promoter and upstream region <strong>of</strong> Dll4 is incapable <strong>of</strong> driving endothelial<br />
expression in mouse or zebrafish. We identified a minimal intronic enhancer <strong>of</strong> 30-bp that completely<br />
recapitulates the early and late developmental arterial-specific expression <strong>of</strong> Dll4. This activity is conserved in<br />
both mice and zebrafish. Mutagenesis <strong>of</strong> the Dll4 minimal enhancer indicates that Notch signaling via an RpbjK<br />
site is dispensable for arterial expression. The presence <strong>of</strong> other candidate transcription factor binding sites,<br />
including Ets sites, suggests potential regulatory mechanisms for arterial expression <strong>of</strong> Dll4. This enhancer does<br />
not recapitulate endogenous Dll4 expression in the post-natal mouse retina, suggesting that Dll4 expression<br />
may be regulated by different pathways during developmental vasculogenesis and post-natal angiogenesis. As<br />
we define the minimal enhancer <strong>of</strong> Dll4, we will be able to identify the transcriptional inputs required to induce<br />
artery-specific gene expression, and thus the minimal instructions required for arteriogenesis.<br />
4.34 Transmembrane protein 2 (tmem2) is required for cardiovascular development<br />
Benjamin M. Hogan, Huijun Chen, Jessica De Angelis, Scott Paterson, Carol Wicking & Kelly A. Smith<br />
Institute for Molecular Bioscience, The <strong>University</strong> <strong>of</strong> Queensland, Brisbane, Queensland 4072, Australia<br />
The endocardium and blood vasculature are both derived from cells <strong>of</strong> the endothelial lineage and share<br />
common genetic regulators. tmem2 was recently identified in zebrafish as a novel regulator <strong>of</strong> cardiac<br />
morphogenesis and we show here that tmem2 is also required for angiogenesis. tmem2 mutants are defective<br />
in intersegmental vessel sprouting for both primary (arterial) and secondary (venous) sprouting. In situ<br />
hybridization analysis <strong>of</strong> arterial and venous markers demonstrated that arterial-venous fate is correctly<br />
specified in tmem2 mutants, supporting the notion that the angiogenesis defect is specific. Tmem2 is a 1390<br />
amino acid protein with a single-pass transmembrane domain (located close to the N-terminus) and computer<br />
prediction places the large C-terminus in the extracellular space. Little else is known about Tmem2 structure or<br />
function. To investigate Tmem2 function, we have generated a novel transgenic tool to watch live protein<br />
dynamics in the developing embryo. Using BAC recombineering, we have generated a Tmem2-Cherry fusion<br />
protein under promoter control that is predicted to recapitulate endogenous expression. This transgene rescues<br />
the mutant phenotypes, demonstrating that we are observing the only functional copy <strong>of</strong> the protein as it<br />
performs its function. Live imaging <strong>of</strong> this line has provided novel insights into Tmem2 protein behavior and<br />
localization during angiogenic sprouting and cardiac morphogenesis. This work introduces a new regulator <strong>of</strong><br />
angiogenesis and describes a novel tool that is providing unique insights into Tmem2 function.<br />
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4.35 Targeted non-invasive occlusion <strong>of</strong> pharyngeal arch arteries during avian cardiac morphogenesis<br />
Stephanie Lindsey, Huseyin C. Yalcin, Akshay Shekhar, Nozomi Nishimura, Chris B. Schaffer, Jonathan T.<br />
Butcher<br />
Department <strong>of</strong> Biomedical Engineering, Cornell <strong>University</strong><br />
The role <strong>of</strong> hemodynamics in outflow tract and pharyngeal arch artery morphogenesis is poorly understood.<br />
Through the use <strong>of</strong> two- photon microscopy guided femtosecond laser pulses, we nucleated and controlled the<br />
growth <strong>of</strong> microbubbles within outflow vessels without disturbing surrounding tissues. These bubbles<br />
temporarily occluded the vessel, during which time a stable occlusion could be formed by ablating the circulating<br />
thrombocytes that accumulated behind the bubble. These clot-like structures then persisted. Using this<br />
approach, we examine the effects <strong>of</strong> PAA vessel occlusions on embryonic viability, hemodynamic<br />
rearrangement, and downstream outflow tract morphogenesis. We determine that occlusion <strong>of</strong> the right IV<br />
pharyngeal arch artery caused lethality in 99.5% <strong>of</strong> embryos by day 6 (HH28/29), while occlusion <strong>of</strong> the left IV<br />
arch artery was significantly less lethal. In each case, blood flow was redistributed to the III and VI PAA and<br />
vessel diameters were altered significantly. With right IV PAA occlusion, the right III PAA artery increased 47%<br />
in diameter while the right VI decreased 100%. Interestingly, ultrasound derived blood velocity did not change in<br />
the right III PAA but significantly increased in the right VI. No changes in diameter or velocity were seen in the<br />
contralateral PAA. When both IV PAA were occluded, both PAA vessels changed in diameter, resulting in an<br />
overall decrease in ouflow lumen area. These results support the hypothesis that PAA hemodynamics is an<br />
important contributor to embryonic development.<br />
4.36 Etv2 regulates endothelial development via activation <strong>of</strong> Sox7<br />
Ann N. Behrens 1 , Claudia Zierold 1 , Yi Ren 1 , Daniel J. Garry 1,2 and Cindy M. Martin 1,2<br />
1 Lillehei Heart Institute, <strong>University</strong> <strong>of</strong> Minnesota, Minneapolis, Minnesota. 2 Department <strong>of</strong> Internal Medicine,<br />
<strong>University</strong> <strong>of</strong> Texas Southwestern Medical Center, Dallas, Texas.<br />
Etv2 has recently been shown to be an important factor for the specification <strong>of</strong> the endocardial/endothelial and<br />
hematopoietic lineages. Transgenic embryos lacking Et2v die between E9.0 and E9.5 due to absence <strong>of</strong> the<br />
endocardial/endothelial lineage and significant disruption <strong>of</strong> hematopoietic lineages. Sox 7, Sox 17 and Sox 18<br />
comprise the Sox-F family which has been shown to be important in the formation <strong>of</strong> endoderm, regulation <strong>of</strong><br />
hematopoietic cells and cardiovascular development. Given that the Etv2 null mice had defects in<br />
cardiovascular and hematopoietic development we questioned whether the Sox F members may be<br />
downstream targets <strong>of</strong> Etv2. Sox 7, Sox 17 and Sox 18 were found to be significantly downregulated in the Etv2<br />
null mice compared to WT littermates at two timepoints (E8.0 and E 8.5). To mechanistically dissect the<br />
regulation <strong>of</strong> this molecular program, we utilized an array <strong>of</strong> molecular biological techniques to demonstrate that<br />
Etv2 is a direct upstream regulator <strong>of</strong> the Sox 7 gene. We generated a doxycycline-inducible Sox7-myc tagged<br />
overexpressing ES cell line. Overexpression <strong>of</strong> Sox7 in engineered EBs resulted in an induction <strong>of</strong> the<br />
endothelial pathway evidenced by an increase in the percentage <strong>of</strong> EB day 6, Flk-1/Cdh5 double positive<br />
endothelial progenitor cells and increased expression <strong>of</strong> Pecam1 and Cdh5 in protein harvested from Sox7<br />
overexpressing EBs. Collectively, these studies support the conclusion that Etv2 is essential for the<br />
endocardial/endothelial lineage and Sox7 is an important factor in this developmental pathway.<br />
Section 5: Coronaries, Epicardium, and Conduction System<br />
S5.1 Ets-1 Regulates the Migration <strong>of</strong> Coronary Vascular Precurors and Coronary Endothelial Cell<br />
Proliferation<br />
Gene H. Kim, Saoirse S. McSharry, Zhiguang Gao, Ankit Bhatia, Judy Earley, and Eric C. Svensson<br />
Department <strong>of</strong> Medicine, The Universtiy <strong>of</strong> <strong>Chicago</strong>, <strong>Chicago</strong>, IL 60637<br />
We have recently described a cardiac developmental defect in mice deficient in the transcription factor Ets-1.<br />
These mice die in the perinatal period with ventricular septal defects and an ectopic focus <strong>of</strong> cartilage in their<br />
outflow tract myocardium. We demonstrated that these defects are the result <strong>of</strong> altered migration and<br />
differentiation <strong>of</strong> the cardiac neural crest due to the loss <strong>of</strong> Ets-1. In this report, we describe an additional role for<br />
Ets-1 during cardiac development. We observed left ventricular systolic dysfunction in Ets-1 -/- mice as measured<br />
by a decrease in fractional shortening (45.3% vs. 22.5%, p
earlier steps in coronary vasculogenesis, we performed whole-mount PCAM staining <strong>of</strong> E12.5 to E14.5 Ets-1 -/-<br />
hearts and found significant attenuation in the development <strong>of</strong> the coronary vascular plexus. The appearance <strong>of</strong><br />
both arterial and venous coronary precursors in the subepicardium was delayed, suggesting a global defect in<br />
the migration <strong>of</strong> these cells. In addition, phospho-histone H3 immunohistochemistry demonstrated a 41%<br />
reduction in the proliferation <strong>of</strong> endothelial cells in Ets-1 -/- hearts at E16.5. Taken together, these results suggest<br />
that Ets-1 plays an important role in both the migration and proliferation <strong>of</strong> coronary endothelial cells during<br />
cardiac development.<br />
S5.2 Wnt signaling in the developing murine epicardium and epicardium-derived-cells<br />
(EPDCs)<br />
Carsten Rudat 1* , Julia Norden 1 , Makoto M. Taketo 2 , Andreas Kispert 1<br />
1 Institute for Molecularbiology, Hannover Medical School, Germany,<br />
2 Department <strong>of</strong> Pharmacology, Kyoto <strong>University</strong> Graduate School <strong>of</strong> Medicine, Japan.<br />
The epicardium is the outermost layer <strong>of</strong> the heart and develops from a mesothelial cluster <strong>of</strong> cells at its venous<br />
pole (the proepicardium). A subset <strong>of</strong> these cells invades the underlying subepicardium and myocardium via an<br />
epithelial-to-mesenchymal-transition (EMT). Besides this cellular contribution additional epicardium-derived<br />
paracrine signals are important for myocardial and coronary vessel development. Previous work suggested a<br />
requirement <strong>of</strong> canonical Wnt signaling in the epicardium for EMT and coronary smooth muscle cell<br />
development. Here, we reinvestigated the role <strong>of</strong> canonical Wnt signaling in the epicardium and epicardiumderived-cells<br />
during embryogenesis, using an alternative conditional genetic approach. In the background <strong>of</strong> a<br />
Tbx18cre-line showing recombination in the proepicardium and its derivatives, ß-catenin (Ctnnb1) loss- and<br />
gain-<strong>of</strong>-function alleles were analyzed. Surprisingly, mice deficient for Ctnnb1 are live born and show neither<br />
impaired coronary artery formation, nor a defective epicardium. The ability <strong>of</strong> epicardium-derived explants to<br />
differentiate into the smooth muscle lineage is maintained. Stabilization <strong>of</strong> Ctnnb1 in the epicardium resulted in<br />
lethality between embryonic days eleven and twelve. Further lineage analysis revealed the formation <strong>of</strong><br />
undifferentiated cell aggregates on the surface <strong>of</strong> the developing heart and impaired EMT. Our data contradicts<br />
previous work, by demonstrating that canonical Wnt signaling in epicardium and EPDCs is dispensable for<br />
coronary artery formation in the developing embryo. Moreover constitutive active Wnt signaling in these cells<br />
leads to early lethality, indicating only a minor physiological role <strong>of</strong> Wnt signaling in the epicardium and<br />
epicarium-derived-cells during development.<br />
S5.3 Determination <strong>of</strong> Cardiac Pacemaker Cell Origins Reveals a Critical Role for Wnt Signaling During<br />
their Cell Fate Specification.<br />
Michael Bressan, Gary Liu, and Takashi Mikawa<br />
<strong>University</strong> <strong>of</strong> California San Francisco, Cardiovascular research Institute<br />
Cardiac pacemaker cells have unique molecular and physiological characteristics that allow them to rapidly and<br />
autonomously generate action potentials. How and when pacemaker cell fate diversifies from adjacent<br />
myocardial cell types and are programmed to attain these specialized features, however, remains poorly<br />
understood. Here we report our fate mapping studies demonstrating that just following gastrulation pacemaker<br />
progenitors reside in the right lateral plate mesoderm posterior to the known heart fields. Our data further<br />
indicate that this pacemaker precursor region overlaps with the expression <strong>of</strong> at least one canonical Wnt,<br />
Wnt8c. This is surprising as canonical Wnts are thought to be inhibitory for myocardial specification during these<br />
stages. In order to test whether Wnt signaling is required to specifically induce pacemaker versus working<br />
myocardial fate, we injected cells expressing the soluble Wnt antagonists, Crescent, directly into the pacemaker<br />
region <strong>of</strong> gast rulating embryos. Ectopic Crescent resulted in both the expansion <strong>of</strong> the transcription factor<br />
NKX2.5 and the down-regulation <strong>of</strong> at least one pacemaker ion channel, HCN4, within the pacemaker<br />
precursors. Conversely, introduction <strong>of</strong> canonical Wnts into the heart field mesoderm was capable <strong>of</strong> inducing<br />
ectopic pacing sites and the formation <strong>of</strong> NKX2.5 negative HCN4 positive myocytes within the looping heart<br />
tube, suggesting a conversion <strong>of</strong> working myocytes into pacemaker-like cells. These data demonstrate that<br />
pacemaker progenitor specification depend on signaling cues unique from working myocardium and that Wnt<br />
signaling, at least in part, supports divergence into the pacemaker lineage. This work is supported in part by<br />
grants from the NIH-NLHBI.<br />
S5.4 A Tbx5-Scn5a Molecular Network Modulates Function <strong>of</strong> the Cardiac Conduction System<br />
David E Arnolds (1), Fang Liu (2), Scott Smemo (1), John P Fahrenbach (1), Gene H Kim (1), Ozanna Burnicka-<br />
Turek (1), Elizabeth M McNally (1), Marcelo M Nobrega (1), Vickas V Patel (2), and Ivan P Moskowitz (1)<br />
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<strong>University</strong> <strong>of</strong> <strong>Chicago</strong>; <strong>Chicago</strong>, IL (1) and <strong>University</strong> <strong>of</strong> Pennsylvania; Philadelphia, PA (2)<br />
Cardiac conduction system (CCS) disease is common with significant morbidity and mortality. Current treatment<br />
options are limited and rational efforts to develop cell-based and regenerative therapies require knowledge <strong>of</strong><br />
the molecular networks that establish and maintain CCS function. Emerging evidence points to key CCS roles<br />
for genes with established roles in heart development. Recent genome wide association studies (GWAS) have<br />
identified numerous loci associated with human CCS function including TBX5 and SCN5A. We hypothesized<br />
that Tbx5, a critical developmental transcription factor, regulates networks required for mature CCS function.<br />
Removal <strong>of</strong> Tbx5 from the mature VCS resulted in severe VCS functional consequences, including loss <strong>of</strong> fast<br />
conduction, arrhythmias, and sudden death. Ventricular contractile function and the VCS fate map remained<br />
unchanged in VCS-specific Tbx5 knockouts. However, key mediators <strong>of</strong> fast conduction including Cx40 and<br />
Nav1.5, encoded by Scn5a, demonstrated Tbx5-dependent expression in the VCS. We identified a Tbx5responsive<br />
enhancer downstream <strong>of</strong> Scn5a sufficient to drive VCS expression in vivo, dependent on canonical<br />
T-box binding sites. Our results establish a direct molecular link between Tbx5 and Scn5a and establish a<br />
hierarchy between human GWAS loci that affects VCS function in the mature CCS, establishing a paradigm for<br />
understanding the molecular pathology <strong>of</strong> VCS disease. Ongoing studies to dissect the regulation <strong>of</strong> Scn5a<br />
expression using a Scn5a-LacZ BAC transgenic reporter system have identified two Scn5 a enhancers sufficient<br />
for establishing the native Scn5a expression pattern. Implications for the role <strong>of</strong> Scn5a and Scn10a in<br />
conduction system function, variability, and arrhythmia susceptibility will be discussed.<br />
S5.5 Mouse cardiac T-box targets reveal an Scn5a/10a enhancer functionally affected by genetic<br />
variation in humans<br />
Phil Barnett #,1 , Malou van den Boogaard 1 , L. Y. Elaine Wong 1 , Federico Tessadori 4 , Martijn L. Bakker 1 , Connie<br />
R. Bezzina 2 , Peter A.C. ‘t Hoen 3 , Jeroen Bakkers 4 , Vincent M. Christ<strong>of</strong>fels #,1<br />
1 Department <strong>of</strong> Anatomy, Embryology and Physiology and 2 Department <strong>of</strong> Experimental Cardiology, Heart Failure Research<br />
Center, Academic Medical Center, <strong>University</strong> <strong>of</strong> Amsterdam, The Netherlands; 3 Center for Human and Clinical Genetics and<br />
Leiden Genome Technology Center, Leiden <strong>University</strong> Medical Center, Leiden, The Netherlands; 3 Cardiac development and<br />
genetics group, Hubrecht Institute for Developmental Biology and Stem Cell Research, Utrecht, The Netherlands. # authors<br />
contributed equally and share correspondance<br />
The contraction pattern <strong>of</strong> the heart relies on the activation and conduction <strong>of</strong> the electrical impulse.<br />
Perturbations <strong>of</strong> cardiac conduction have been associated with congenital and acquired arrhythmias and cardiac<br />
arrest. The pattern <strong>of</strong> conduction depends on the regulation <strong>of</strong> heterogeneous gene expression by key<br />
transcription factors and transcriptional enhancers. Here, we assessed the genome-wide occupation <strong>of</strong><br />
conduction system regulating transcription factors Tbx3, Nkx2-5 and Gata4 and <strong>of</strong> enhancer-associated coactivator<br />
p300 in the mouse heart, uncovering cardiac enhancers throughout the genome. Many <strong>of</strong> the<br />
enhancers co-localize with ion channel genes repressed by Tbx3, including the clustered sodium channel genes<br />
Scn5a, essential for cardiac function, and Scn10a. We identify two enhancers in the Scn5a/Scn10a locus, which<br />
are regulated by Tbx3 and its family member and activator Tbx5, and are functionally conserved in humans. We<br />
provide evidence that a single-nucleotide po lymorphism in the SCN10A enhancer, associated with alterations in<br />
cardiac conduction patterns in humans, disrupts Tbx3/5 binding and reduces the cardiac activity <strong>of</strong> the enhancer<br />
in vivo. Thus, the identification <strong>of</strong> key regulatory elements for cardiac conduction helps to explain how genetic<br />
variants in non-coding regulatory DNA sequences influence the regulation <strong>of</strong> cardiac conduction and the<br />
predisposition for cardiac arrhythmias.<br />
S5.6 Activation <strong>of</strong> voltage-dependent anion channel 2 suppresses Ca 2+ -induced cardiac arrhythmia<br />
Hirohito Shimizu 1 , Johann Schredelseker 1 , Jie Huang 1 , Kui Lu 2 , Sarah Franklin 3 , Kevin Wang 1 , Thomas<br />
Vondriska 3 , Joshua Goldhaber 4 , Ohyun Kwon 2 , Jau-Nian Chen 1<br />
1 Department <strong>of</strong> Molecular, Cell and Developmental Biology, 2 Department <strong>of</strong> Chemistry and Biochemistry,<br />
3 Department <strong>of</strong> Anesthesiology, <strong>University</strong> <strong>of</strong> California, Los Angeles and 4 Cedars-Sinai Heart Institute, Los<br />
Angeles<br />
While a role <strong>of</strong> Ca 2+ in establishing embryonic cardiac rhythmicity has been proposed, the underlying<br />
mechanisms have yet to be fully explored. The zebrafish tremblor (tre) mutant embryo lacks functional cardiac<br />
Na + /Ca 2+ exchanger, resulting in Ca 2+ extrusion defects, abnormal Ca 2+ transients and unsynchronized cardiac<br />
contractions. We therefore use this mutant as an animal model to dissect the molecular network essential for<br />
controlling Ca 2+ homeostasis in the heart. From a chemical suppressor screen, we identified a synthetic<br />
compound named efsevin, which effectively restores synchronized cardiac contractions in tre embryos. In<br />
addition, efsevin suppresses arrhythmogenic Ca 2+ waves by accelerating the decay phase <strong>of</strong> Ca 2+ sparks in<br />
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murine cardiomyocytes, demonstrating that efsevin modulates cardiac Ca 2+ handling. Through a biochemical<br />
pull-down assay, we identified a direct interaction between efsevin and the mitochondrial outer membrane<br />
protein VDAC2. Overexpression <strong>of</strong> VDAC2 restores synchronized cardiac contractions in tre embryos and<br />
knock-down <strong>of</strong> VDAC2 attenuates the rescue effect <strong>of</strong> efsevin, indicating that efsevin modulates Ca 2+ handling<br />
by enhancing the activity <strong>of</strong> VDAC2. Furthermore, overexpression <strong>of</strong> mitochondrial inner membrane Ca 2+ uptake<br />
proteins restores cardiac contractions in tre embryos. This effect is further enhanced when VDAC2 is coexpressed,<br />
and is abolished in tre/vdac2 double deficient embryos. Taken together, our data reveal an essential<br />
role for mitochondria in regulating cardiac Ca 2+ homeostasis and rhythmicity. Our findings also establish VDAC2<br />
as a critical gate for mitochondrial Ca 2+ uptake and efsevin as a potential therapeutic agent for cardiac<br />
arrhythmia.<br />
5.7 The BMP regulator BMPER is necessary for normal coronary artery formation<br />
Laura Dyer and Cam Patterson<br />
McAllister Heart Institute, <strong>University</strong> <strong>of</strong> North Carolina at Chapel Hill<br />
Connection <strong>of</strong> the coronary vasculature to the aorta is one <strong>of</strong> the last essential steps <strong>of</strong> cardiac development.<br />
However, little is known about the signaling events that promote normal coronary artery formation. The bone<br />
morphogenetic protein (BMP) signaling pathway regulates multiple aspects <strong>of</strong> endothelial cell biology but has<br />
not been specifically implicated in coronary vascular development. BMP signaling is tightly regulated by<br />
numerous factors, including BMP-binding endothelial cell precursor-derived regulator (BMPER), which can both<br />
promote and repress BMP signaling activity. Analysis <strong>of</strong> the BMPER-/- mouse indicated that this deficiency<br />
leads to embryonic death, and in the embryonic heart, BMPER expression is limited to the endothelial cells and<br />
the endothelial-derived cushions, suggesting that coronary vascular defects may be present. Histological<br />
analysis <strong>of</strong> BMPER-/- embryos at embryonic day 16.5 revealed that the coronary arteries were either atretic or<br />
connected distal to the semilunar valves. However, analysis <strong>of</strong> earlier embryonic stages showed that the<br />
coronary plexus began differentiating normally and that apoptosis and cell proliferation were unaffected in<br />
BMPER-/- embryos. In vitro tubulogenesis assays showed that isolated BMPER-/- endothelial cells had impaired<br />
tube formation compared to wild-type endothelial cells. Together, these results indicate that BMPER-regulated<br />
BMP signaling is critical for the migration <strong>of</strong> coronary endothelial cells and normal coronary artery development.<br />
5.8 Epicardial chemokine signaling influences ventricular wall proliferation and coronary<br />
vasculogenesis<br />
Susana Cavallero, Hua Shen, Peng Li, Henry M. Sucov.<br />
Broad Center for Regenerative Medicine and Stem Cell Research, Keck School <strong>of</strong> Medicine, <strong>University</strong> <strong>of</strong><br />
Southern California, Los Angeles, CA.<br />
During heart development, the epicardium secretes soluble factors that direct the morphological organization <strong>of</strong><br />
the underlying myocardial wall. Our previous study established that insulin-like growth factor 2 (IGF2) is the<br />
primary mitogen expressed by the epicardium that controls embryonic ventricular cardiomyocyte proliferation.<br />
We found that CXCL12 (stromal derived factor-1) is an additional factor expressed by epicardial cells in vitro<br />
and by the epicardium in vivo. Cultured primary cardiomyocytes treated with epicardial conditioned media or in<br />
coculture with epicardial cells show a proliferative response that is partially blocked by AG1024 (an IGFR<br />
inhibitor) or AMD3100 (a CXCR4 inhibitor), and almost completely abolished by both compounds together. To<br />
date, a functional role for CXCL12 signaling in heart development has not been examined. Our results indicate<br />
that both global Cxcl12 null and cardiac-specific (Nkx2.5Cre) Cxcr4 null embryos show a moderate deficiency in<br />
compact zone proliferation. Embryos with combined loss <strong>of</strong> IGF2 and CXCL12 signaling exhibit pronounced<br />
edema, suggesting that epicardial IGF2 and CXCL12 together promote mitogenic signaling needed for<br />
cardiomyocyte proliferation and compact zone expansion. Because CXCL12 is a chemokine that has known<br />
effects on vascular cells, we also examined coronary vessel formation in embryos with disrupted CXCL12<br />
signaling. We observed expanded superficial vessels and a reduced number <strong>of</strong> intramyocardial arteries. Our<br />
results indicate that the epicardium is a source <strong>of</strong> secreted CXCL12 that participates with the major mitogen<br />
IGF2 to induce cardiomyocyte proliferation and compact zone expansion, and also acts on endothelial cells to<br />
support formation <strong>of</strong> the coronary vasculature.<br />
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5.9 The role <strong>of</strong> Pod1/Tcf21 in epicardium-derived cells during cardiac development and fibrosis<br />
Caitlin M. Braitsch 1 , Michelle D. Combs 1 , Susan E. Quaggin 2 , and Katherine E. Yutzey 1 .<br />
1 Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA; 2 <strong>University</strong> <strong>of</strong> Toronto, Toronto, Ontario,<br />
Canada.<br />
During cardiac development, epicardium-derived cells (EPDCs) differentiate into fibroblasts and vascular<br />
smooth muscle (SM) cells. In the adult heart, EPDCs are reactivated upon cardiac injury. Immun<strong>of</strong>luorescence<br />
studies demonstrate that the transcription factors (TF) Pod1/Tcf21, WT1, Tbx18, and NFATC1 are expressed<br />
heterogeneously in EPDCs in chicken and mouse embryonic hearts. Expression <strong>of</strong> Pod1 and WT1, but not<br />
Tbx18 or NFATC1, is activated with all-trans-retinoic acid (RA) treatment <strong>of</strong> isolated avian EPDCs. In intact E7<br />
chicken hearts, RA signaling is required for full Pod1 expression and RA treatment inhibits SM differentiation.<br />
The requirements for Pod1 in differentiation <strong>of</strong> EPDCs in the developing heart were examined in mice lacking<br />
Pod1. Loss <strong>of</strong> Pod1 in mice leads to epicardial blistering, increased SM differentiation on the surface <strong>of</strong> the<br />
heart, and a paucity <strong>of</strong> interstitial fibroblasts, with neonatal lethality. On the surface <strong>of</strong> the myocardium,<br />
expression <strong>of</strong> multiple SM markers is increased in Pod1-deficient EPDCs, demonstrating premature SM<br />
differentiation. Together, these data demonstrate a critical role for Pod1 in controlling EPDC differentiation into<br />
SM during cardiac development. In adult hearts, cardiac injury leads to reactivation <strong>of</strong> fetal genes including<br />
WT1, Tbx18, and RALDH2 in EPDCs. Initial studies demonstrate that Pod1/Tcf21 expression is increased in<br />
mouse hearts subjected to myocardial infarction, transverse aortic constriction, or chronic isoproterenol infusion.<br />
Additional data will be presented describing Pod1/Tcf21 expression in mouse models <strong>of</strong> cardiac fibrosis.<br />
Together this work provides evidence for conserved Pod1/Tcf21-related regulatory mechanisms in EPDCs<br />
during cardiac development and fibrotic disease.<br />
5.10 Epicardial GATA factors regulate coronary endothelial migration via Sonic hedgehog signaling.<br />
Kurt D. Kolander, Mary L. Holtz, and Ravi P. Misra<br />
Department <strong>of</strong> Biochemistry, Medical College <strong>of</strong> Wisconsin, Milwaukee, WI<br />
Early coronary vasculogenesis is dependent on the epicardium as a source <strong>of</strong> progenitor cells and regulatory<br />
signals. GATA transcription factors have been shown to be required for the formation <strong>of</strong> the proepicardium, a<br />
group <strong>of</strong> cells that give rise to the epicardium This suggests GATA factors may play a role in epicardial function.<br />
To address whether epicardial GATA-4 and GATA-6 may have a role in early coronary vascular development,<br />
we utilized a Wilm’s Tumor-1 promoter-driven Cre recombinase construct to conditionally knockout GATA-4 and<br />
GATA-6 in the epicardium. We found the combined loss <strong>of</strong> GATA-4 and GATA-6 resulted in embryonic lethality<br />
at E15.5, an age at which various coronary vascular defects commonly lead to lethality. Immun<strong>of</strong>luorescent<br />
imaging at E13.5 and E14.5 indicated an 80-90% decrease in sub-epicardial endothelial cells, which are<br />
required for coronary plexus formation. This led us to hypothesize that the decreased number <strong>of</strong> sub-epicardial<br />
endothelial cells is due to a loss <strong>of</strong> epicardial signaling to early endothelial or progenitor cells. Sonic hedgehog is<br />
an epicardial signaling factor required for the migration <strong>of</strong> sub-epicardial endothelial cells. Therefore, we<br />
immun<strong>of</strong>luorescently stained for Sonic hedgehog and found its expression was lost in the sub-epicardial space<br />
<strong>of</strong> the conditional knockout. These results are consistent with a novel role for epicardial GATA factors in<br />
coronary vascular development through regulation <strong>of</strong> a Sonic hedgehog signaling pathway<br />
5.11 Coordinated Regulation <strong>of</strong> Pacemaker Ion Channels during SAN Differentiation<br />
Gary Liu, Michael Bressan, Takashi Mikawa<br />
<strong>University</strong> <strong>of</strong> California San Francisco, Cardiovascular research Institute<br />
Cardiac pacemaker cells (CPCs) rhythmically generate electrical signals that dictate the timing <strong>of</strong> heart<br />
contractions. While the ion currents responsible for pacemaker action potentials (APs) have been thoroughly<br />
investigated, the developmental mechanisms regulating the underlying channels and pumps remain poorly<br />
understood. We have previously reported that CPC fate is specified in the mesoderm posterior to the heart field<br />
2 full days before they first become electrically active within the right inflow. To determine the underlying<br />
mechanisms for this lag between fate specification and differentiation, we monitored the expression <strong>of</strong> ion<br />
handling genes necessary for CPC APs during this developmental window. Here we report that HCN4, Serca2,<br />
NCX, and RYR2 are all absent from CPC precursors, but then become up-regulated broadly across the right<br />
inflow as the CPCs first begin pacing the heart. To determine if this coordinated upregulation is dependent on<br />
known CPC transcriptional programs, we examined the expression <strong>of</strong> T-box genes Tbx3, Tbx5, and Tbx18.<br />
Although detectable in the inflow, their expression patterns did not match that <strong>of</strong> the CPC ion channels nor were<br />
any expression pr<strong>of</strong>iles confined to the pacemaking site. This data suggest that the molecular components<br />
127
necessary for CPC AP generation are regulated in a coordinated manner and their upregulation dictates the<br />
timing <strong>of</strong> CPC electrical activation. Additionally, the site <strong>of</strong> action potential production cannot be demarcated<br />
solely by the above factors, indicating that further transcriptional and/or signaling mechanism(s) may be<br />
required. Supported by NIHR01HL112268.<br />
5.12 Cardiac Specific Expression <strong>of</strong> FOG-2 Induces Atrial Fibrillation<br />
Michael Broman, Harold E. Olivey, Gene H. Kim, Saoirse S. McSharry, Eric C. Svensson<br />
Department <strong>of</strong> Medicine, The <strong>University</strong> <strong>of</strong> <strong>Chicago</strong>, <strong>Chicago</strong>, IL<br />
Atrial fibrillation is a common cardiac arrhythmia associated with significant worldwide morbidity and mortality.<br />
Although many factors, including myocyte electrical remodeling, atrial contractile dysfunction, and structural<br />
remodeling have been linked to atrial fibrillation, the molecular causes are still unclear. Previous work has<br />
demonstrated that FOG-2 (Friend <strong>of</strong> GATA-2), a transcriptional co-repressor important in early heart<br />
development, is overexpressed in the hearts <strong>of</strong> patients with heart failure who are prone to developing atrial<br />
fibrillation. We have developed a novel genetic model <strong>of</strong> inducible, cardiac specific FOG-2 expression in mice<br />
via a doxycycline-inducible transgenic system. In these mice, we find an approximately 5-7 fold increase in<br />
FOG-2 protein and mRNA levels in the atrial tissue <strong>of</strong> these animals following gene induction. These animals<br />
have a high incidence <strong>of</strong> spontaneous and sustained atrial fibrillation (approximately 80%), with the remainder <strong>of</strong><br />
these animals having easily induced atrial fibrillation by catheter-based pacing <strong>of</strong> the right atrium. The atrial<br />
fibrillation precedes any left ventricular systolic dysfunction, valvular disease, or elevated preload, indicating a<br />
primary atrial mechanism. Because FOG-2 is known to inhibit the expression <strong>of</strong> multiple cardiac-specific genes,<br />
we performed a microarray-based gene expression analysis on atrial tissue. The results <strong>of</strong> this microarray<br />
suggested that the expression <strong>of</strong> multiple potassium channel subunit proteins (KCNJ5, KCND2 and KCNE1) are<br />
downregulated, and these results were further confirmed by quantitative RT-PCR. These data suggests that<br />
FOG-2 upregulation leads to the downregulation <strong>of</strong> cardiac potassium channels, thus promoting the<br />
development <strong>of</strong> atrial fibrillation.<br />
5.13 Localization and lineage analysis <strong>of</strong> the sinoatrial node precursor cells within the posterior<br />
Secondary Heart Field in the mouse<br />
Mª Magdalena Molero, Diego Franco, Amelia Aránega, Jorge N Domínguez.<br />
Cardiovascular Research Group. Department <strong>of</strong> Experimental Biology, Faculty <strong>of</strong> Experimental Sciences,<br />
<strong>University</strong> <strong>of</strong> Jaén, Jaén, Spain.<br />
The early heart forms from two mesodermal cell populations, the First and Second Heart Fields (F&SHF). The<br />
FHF forms the cardiac crescent (E7.5 in mouse). The SHF lies dorsally and medially, in pharyngeal mesoderm,<br />
and is defined by islet1 expression. The left ventricle (LV) derives exclusively from FHF whereas the distal<br />
outflow tract (OFT) is SHF derived. The proximal OFT and RV are predominantly SHF derived, and the atria are<br />
<strong>of</strong> mixed F&SHF origin. A sub-portion <strong>of</strong> the SHF expresses Fgf10, as shown by the 1V-24 lacZ reporter, which<br />
labels the anterior HF (AHF). The AHF is fated to contribute to the OFT/RV but not the inflow/atria. Using dyelabeling<br />
injections at the posterior portion <strong>of</strong> the SHF (pSHF), which is characterized by Islet1+/1v-24-<br />
expression, we have demonstrated that IF region arise from pSHF (see Galli et al, 2008) and also<br />
atrioventricular canal (AVC) and OFT are pSHF-derived cardiac regions (Domínguez et al, <strong>2012</strong>; submitted).<br />
Lineage studies using islet1-cre and islet1-mer-cre-mer mice crossed with the ROSA26R, have described that<br />
sinoatrial node (SAN) and a central region <strong>of</strong> the atrioventricular node (AVN) are derived from islet1 precursor<br />
cells (SHF) (Moretti et al, 2006; Sun et al, 2007), whereas it is assume that ventricular conduction system (His-<br />
Purkinje system) is a non-SHF derived. In this regard, an accurate lineage analysis <strong>of</strong> SHF-islet1 cells<br />
contribution to the distinct components <strong>of</strong> the cardiac conduction system (CCS) is missing. Moreover, the<br />
localization <strong>of</strong> precursor cells forming the SAN and AVN central region from the SHF is unknown. Base on our<br />
previous data, we are interesting now to determine whether SAN and AVN islet1+ precursor cells are placed at<br />
the pSHF and, moreover, whether islet1+ progenitor cells participate in His-Purkinje system development during<br />
cardiogenesis. We are labelling small groups <strong>of</strong> cells <strong>of</strong> 4-6 somites mouse embryos, throughout the pSHF<br />
using fluorescent dyes. After that, we culture the embryos for 48 hours and then we perform<br />
immunohistochemistry in whole mount against HCN4, a well-known SAN molecular marker. Preliminary results<br />
evidence that, in most cases, right and left pSHF derived cells populate the right and left atria <strong>of</strong> the developing<br />
heart (HCN4-), respectively. However, some dye-labelled cells were placed at the right atria-sinus venous<br />
junction, where HCN4 is expressed and define the putative SAN precursor. These results would suggest that<br />
islet1+ progenitors cells localized at the pSHF participate in the developing <strong>of</strong> both, the working and conduction<br />
128
system myocardium, and open new ways to explore the putative SHF origin <strong>of</strong> the distinct cardiac conduction<br />
system elements.<br />
5.14 Emergence <strong>of</strong> novel myocardial subtypes during dynamic patterning <strong>of</strong> impulse conduction<br />
pathways at the pacemaker-atrial junction<br />
Jonathan D. Louie, Michael Bressan, Takashi Mikawa<br />
<strong>University</strong> <strong>of</strong> California San Francisco, Cardiovascular research Institute<br />
Cardiac pacemaker cells <strong>of</strong> the sinoatrial node initiate electrical impulses that drive rhythmic contraction <strong>of</strong> the<br />
heart. In the mature heart, pacemaker cells are protected from electrical suppression imposed by adjacent<br />
working myocardium by a zone exhibiting poor electrical coupling. However, little is known about the<br />
developmental mechanisms that pattern the low conductance zone that is critical for proper pacemaker<br />
functions. Here we report that pacemaker precursors differentiate within the ventral surface <strong>of</strong> the right inflow<br />
during heart looping and begin to produce pacemaker action potentials (AP). At this stage, APs propagate along<br />
the ventral surface directly into the forming atria and no low conductance zone is evident at or around the<br />
pacemaker cells. As septation initiates, a dynamic shift in propagation pattern occurs as a conduction delay<br />
forms at the junction <strong>of</strong> the right inflow and the atria, and a novel dorsal propagation pathway becomes the<br />
dominant route. Importantly, the cells that compose this zone uniquely express slow skeletal myosin heavy<br />
chain. These junctional myocytes are distinguishable from pacemaker cells that co-express Ventricular myosin<br />
heavy chain1 (VMHC1) and Atrial myosin heavy chain 1(AMHC1), and atrial myocytes that express AHMC1<br />
alone. These data demonstrate dynamic patterning <strong>of</strong> conduction properties at the pacemaker-atrial junction<br />
and suggest that these properties may be related to the emergence <strong>of</strong> myocyte subtypes with distinct<br />
transcriptional programs. Supported by R01 HL093566.<br />
5.15 Endocardium-derived Follistatin-like-1 determines atrioventricular conduction.<br />
Marc Sylva, Bas Boukens, Quinn Gunst, Antoon Moorman, Ruben Coronel*, Vincent Christ<strong>of</strong>fels, Maurice van<br />
den H<strong>of</strong>f<br />
Department <strong>of</strong> Anatomy, Embryology & Physiology, Department <strong>of</strong> Cardiology*, Heart Failure Research Center,<br />
Meibergdreef 15, 1105AZ Amsterdam, The Netherlands<br />
In the adult heart the atrioventricular (AV) node generates the delay between atrial and ventricular activation.<br />
During development the AV delay is generated by the slowly conducting AV canal myocardium, from which the<br />
demarcated AV node forms. How the AV canal myocardium becomes (1) confined to an AV-node and (2)<br />
insulated by connective tissue, is largely unknown. Follistain-like 1 (Fstl1) is a secreted protein that potentially<br />
regulates cardiac development by binding TGFβ super-family members, like BMPs. Fstl1 is expressed from<br />
gastrulation onwards in various organs including the heart, particularly in the endocardially-derived cells that<br />
contribute, to the developing AV valves and insulating plane. Inactivation <strong>of</strong> Fstl1 in mouse results in post-natal<br />
death due to respiratory defects. To determine the role <strong>of</strong> endocardial-derived Fstl1 in the formation <strong>of</strong> the AV<br />
node, we generated endocardium-specific Fstl1 KO mice and studied the structure and function <strong>of</strong> the AV<br />
junction. Endocardium-specific, but not myocardium-specific, deletion <strong>of</strong> Fstl1 resulted in PR prolongation in<br />
neonates, underscoring the specific role <strong>of</strong> endocardium-secreted Fstl1 in AV node function. Within four weeks<br />
after birth these mice continued to have a prolonged PR interval and died. In these mice, primitive AV canal<br />
myocardium, marked by Hcn4, was still present below the valves. BMP reporter assays revealed that Fstl1 is<br />
capable to inhibit BMP signalling, albeit only at low concentrations. Taken together our data suggests that<br />
endocardium-derived Fstl1 inhibits BMP at low concentration, which is required for normal development <strong>of</strong> the<br />
AV junction including the AV node.<br />
5.16 Assessment <strong>of</strong> the genome-wide occupation <strong>of</strong> cardiac transcription factors reveals two<br />
conserved T-box factor-regulated enhancers for the expression patterns <strong>of</strong> Scn5a and Scn10a<br />
Malou van den Boogaard 1 , L. Y. Elaine Wong 1 , Federico Tessadori 4 , Connie R. Bezzina 2 , Peter A.C. ‘t Hoen 3 ,<br />
Jeroen Bakkers 4 , Vincent M. Christ<strong>of</strong>fels #,1 & Phil Barnett #,1<br />
1 Department <strong>of</strong> Anatomy, Embryology and Physiology and 2 Department <strong>of</strong> Experimental Cardiology, Heart Failure Research<br />
Center, Academic Medical Center, <strong>University</strong> <strong>of</strong> Amsterdam, The Netherlands; 3 Center for Human and Clinical Genetics and<br />
Leiden Genome Technology Center, Leiden <strong>University</strong> Medical Center, Leiden, The Netherlands; 3 Cardiac development and<br />
genetics group, Hubrecht Institute for Developmental Biology and Stem Cell Research, Utrecht, The Netherlands. # authors<br />
contributed equally.<br />
Repression <strong>of</strong> the working myocardial phenotype is essential for the development <strong>of</strong> the nodal components <strong>of</strong><br />
the conduction system. Tbx3 plays a central role in this process, regulating the expression <strong>of</strong> many key<br />
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conduction genes and thereby defining the electrophysiological phenotype <strong>of</strong> the sinus and atrioventricular<br />
nodes. The specific down-regulation <strong>of</strong> connexins and ion channels that are associated with rapid conduction is<br />
essential for the correct delay and thus function <strong>of</strong> the atrioventricular node. The cardiac sodium channel<br />
protein, Scn5a, is an essential mediator <strong>of</strong> cardiomyocyte polarization and plays a central role in rapid<br />
conduction <strong>of</strong> the electrical impulse. Mutations in Scn5a give rise to a wide spectrum <strong>of</strong> conduction defects and<br />
have been associated with an increased risk <strong>of</strong> ventricular fibrillation. Loss <strong>of</strong> Tbx3 expression results in the<br />
ectopic expression <strong>of</strong> Scn5a in the nodal components <strong>of</strong> the conduction system and over-expression <strong>of</strong> Tbx3 in<br />
working myocardium reveals its down-regulation. Using a genome wide ChIP-seq approach we have identified<br />
functional binding sites for Tbx3 and cardiac partners <strong>of</strong> Tbx3, Gata4 and Nkx2-5, across the mouse genome.<br />
Combining this data with other ChIP-seq data and microarray data we identified a locus encompassing both<br />
Scn5a and Scn10a, possessing 2 putative enhancers. In vivo enhancer screening confirmed the ability <strong>of</strong> these<br />
DNA elements to drive LacZ expression in a similar patterns to that <strong>of</strong> endogenous Scn5a/10a and that these<br />
elements are conserved in humans. We show that a recently identified GWAS SNP connected to alterations in<br />
AV-conduction and positioned directly under a ChIP-seq peak <strong>of</strong> one <strong>of</strong> the enhancers, appears to attenuate Tbox<br />
binding to this enhancer, thus interfering with transcription regulated by this enhancer. The effect <strong>of</strong> this<br />
attenuation is a loss <strong>of</strong> correct ventricular expression, an assessment based on in vivo analyses <strong>of</strong> the human<br />
enhancer in both fish and mouse model systems.<br />
5.17 Exclusion <strong>of</strong> Endothelin Signaling is Required for Slow Conducting Fate in the Atrioventricular<br />
Junction<br />
PoAn Brian Yang, Mike Bressan, Alicia Navetta, Takashi Mikawa<br />
<strong>University</strong> <strong>of</strong> California San Francisco<br />
Efficient blood flow requires sequential and unidirectional contraction <strong>of</strong> cardiac chambers. The Atrioventricular<br />
junction (AVJ) delays action potential propagation between the developing atria and ventricles allowing for atrial<br />
contraction to complete before ventricular activation. This delay represents the first detectable heterogeneity in<br />
conduction velocity in the heart. The mechanisms responsible for establishing this heterogeneity, however, are<br />
poorly understood. Previously our group has demonstrated that in the embryonic chick ventricle, endothelin is<br />
capable <strong>of</strong> inducing the expression <strong>of</strong> a high conductance gap junction connexin 40 (Cx40) which is present in<br />
the fast conducting Purkinje fiber network but absent from the AVJ. We therefore sought to address whether<br />
lack <strong>of</strong> endothelin signaling in the AVJ is required for this region to escape a fast conducting fate. Here we<br />
report that while one <strong>of</strong> the ET receptors, ETB, is excluded from the developing AVJ, all oth er Endothelin<br />
signaling components, including ppET1, ECE1 and ETA are expressed there. Furthermore, the receptor ETA<br />
can function in the AVJ as exogenous endothelin ectopically induced both the phosphorylation <strong>of</strong> a downstream<br />
endothelin signaling component, Erk, and Cx40 expression at the AVJ. These results demonstrate that AVJ<br />
cells can respond to endothelin signaling converting them from slow conducting cells into fast conducting cells<br />
as in the ventricles. The data also suggest that there must be a mechanism that excludes endothelin signaling<br />
from this region thereby allowing AVJ cells to escape the fast conducting cell fate. Supported by NIH-R01<br />
HL093566, Cardiovascular Diversity Research Supplement, and CIRM.<br />
5.18 Pitx2 prevents the onset <strong>of</strong> an arrhythmogenic program in the mediastinal myocardium:<br />
implications for adult heart function<br />
Grazia Ammirabile, Giulia Rainato and Marina Campione<br />
CNR Institute <strong>of</strong> Neurosciences, Department <strong>of</strong> Biomedical Sciences, <strong>University</strong> <strong>of</strong> Padova, Italy.<br />
Genome wide study populations in humans have indicated PITX2 as a candidate susceptibility gene for atrial<br />
arrhythmias, also confirmed by functional studies in adult Pitx2 heterozygous mice. Arrhythmogenic foci mostly<br />
originate in the pulmonary veins (PV) or in left sinus venosus (SV) derived structures, such as the coronary<br />
sinus or the left superior caval vein; the posterior left atrial wall is additionally critical both for initiation and<br />
maintenance <strong>of</strong> atrial fibrillation. We have previously shown that Pitx2 is required in the SV myocardium to<br />
progressively confine its molecular and functional pacemaker properties exclusively to the right SAN.<br />
We now present the role <strong>of</strong> Pitx2 in the mediastinal myocardium, a Cx40 positive/ANF negative region<br />
comprising the PV myocardium, the primary and secondary interatrial atrial septum (IAS) and left dorsal atrial<br />
wall. Pitx2 is expressed in the left mediastinal region and the derived mediastinal myocardium. We show here<br />
that deletion <strong>of</strong> Pitx2 from cardiomyogenesis onset leads to a reduced extension <strong>of</strong> the PV myocardium and to<br />
its complete reprogramming towards a nodal-conductive type molecular pr<strong>of</strong>ile. Additionally, in the Pitx2<br />
myocardial mutants the dorsal atrial wall and the IAS express ANF, which is molecular substrate for AF.<br />
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Altogether, the nature <strong>of</strong> Pitx2 molecular targets and their topological localization provide support for an antiarrhytmogenic<br />
role <strong>of</strong> the gene at the left venous pole <strong>of</strong> the heart.<br />
5.19 Islet-1 expression identifies the sinoatrial pacemaker <strong>of</strong> the adult zebrafish heart<br />
Federico Tessadori 1 , Silja Burkhard 1 , Henk van Weerd 2 , Arie Verkerk 2 , Emma de Pater 1 , Bas Boukens 2 , Vincent<br />
Christ<strong>of</strong>fels 2 and Jeroen Bakkers 1<br />
1 Hubrecht Institute and <strong>University</strong> Medical Center Utrecht, Utrecht, the Netherlands; 2 Heart Failure Research<br />
Center, Academic Medical Center, <strong>University</strong> <strong>of</strong> Amsterdam, Amsterdam, the Netherlands.<br />
Cardiac development and function are strikingly conserved amongst animal classes. Although the heart’s shape,<br />
relative size and organization may vary, its rhythmic contractions remain crucial for correct cardiac function. In<br />
higher vertebrates such as mammals, specialized pacemaker cells controlling the rhythmicity <strong>of</strong> cardiac<br />
contractions are localized in an anatomically identifiable structure <strong>of</strong> myocardial origin, the sinoatrial node<br />
(SAN). The SAN’s anatomical and functional identification dates back to the early 20th century, before the<br />
availability <strong>of</strong> any molecular marker. In lower vertebrates, however, although cardiac contractions are known to<br />
originate at the venous pole, a localization that is developmentally conserved, isolation <strong>of</strong> such structure has<br />
remained elusive. Here we show that zebrafish embryos lacking the LIM/homeodomain-containing transcription<br />
factor Islet-1 (Isl1) display cardiac rhythmicity defects related to pacemaker dysfunction. Moreover, 3D<br />
reconstructions <strong>of</strong> gene expression patterns in the embryonic and adult zebrafish heart enabled us to uncover a<br />
previously unidentified, ring-shaped region <strong>of</strong> interconnected isl1+ cells in the hcn4+ tbx2b+ nppa- myocardium<br />
at the sinoatrial junction. Using a tg(Isl1BAC:GalFF; UAS:GFP) reporter line made in our laboratory, we<br />
demonstrated by optical mapping <strong>of</strong> action potentials and electrophysiological characterization <strong>of</strong> single isl1+<br />
myocytes that the isl1-expressing cells harbor pacemaker activity. Altogether, our data allow us to establish that<br />
(i) isl1 is the first identified molecular marker for pacemaker function <strong>of</strong> the zebrafish heart and (ii) the functional<br />
pacemaker <strong>of</strong> the zebrafish is organized as a ring at the sinoatrial junction.<br />
5.20 Phenotypic analysis <strong>of</strong> the cardiac conduction system in the absence <strong>of</strong> Tbx1<br />
Beyer S*, Sedmera D°, Kelly RG* and Miquerol L*<br />
* Institute <strong>of</strong> Developmental Biology <strong>of</strong> Marseille-Luminy, CNRS UMR6216, Université de la Méditerranée,<br />
Marseille, France.<br />
° Charles <strong>University</strong> and Institute <strong>of</strong> Physiology, Academy <strong>of</strong> Sciences <strong>of</strong> the Czech Republic, Prague, Czech<br />
Republic<br />
The ventricular conduction system (VCS) is responsible for the rapid propagation <strong>of</strong> electrical activity in the<br />
ventricles in order to synchronize cardiac contraction. The VCS is composed <strong>of</strong> specialized cardiomyocytes<br />
forming the electrical wiring <strong>of</strong> the ventricles including the His bundle, the right (RBB) and left (LBB) bundle<br />
branches and the peripheral Purkinje fibers (PF). TBX1 is a major candidate gene for DiGeorge syndrome,<br />
characterized by crani<strong>of</strong>acial defects and cardiac malformations. Several cardiac defects mimicking the human<br />
syndrome can be observed in Tbx1 null mouse embryos such as common arterial trunk and ventricular septal<br />
defect. At E18.5, Tbx1 null hearts present a distinct phenotype with a non-compact His bundle and absence <strong>of</strong><br />
proximal RBB. Electrical activation maps <strong>of</strong> Tbx1 mutant hearts revealed a left activation <strong>of</strong> ventricles while two<br />
breakthroughs in the right and left ventricles are observed in control hearts. This study revealed that the Tbx1<br />
mutants display a RBB block. At E14.5, the His bundle is positioned more anteriorly in Tbx1 -/- hearts but no<br />
significative differences were observed by optical mapping in these hearts in accordance with the important<br />
network <strong>of</strong> trabeculae present in the right ventricle. This is in accordance with the incomplete insulation between<br />
atria and ventricule at this stage and the presence <strong>of</strong> embryonic electrical pathway at the atrioventricular<br />
junction. Altogether, these data demonstrate that the absence <strong>of</strong> the RBB underlie a RBB block. This<br />
phenotype may occur during fetal stages. To conclude, our data strongly suggest that a defect in cardiac<br />
morphogenesis may be at the origin <strong>of</strong> conduction defect suggesting that a correct development <strong>of</strong> the VCS is<br />
important to acquire a functional heart.<br />
5.21 Epicardial Cells lacking Tgfbr3 have Dysregulated NFkB Signaling<br />
Daniel M. DeLaughter 1 , Cyndi Hill 2 , H. Scott Baldwin 1, 3 , J. G. Seidman 4 , Joey V. Barnett 2<br />
1 Department <strong>of</strong> Cell and Developmental Biology, Vanderbilt <strong>University</strong> Medical Center, Nashville, TN, USA.<br />
2 Department <strong>of</strong> Pharmacology, Vanderbilt <strong>University</strong> Medical Center, Nashville, TN, USA. 3 Department <strong>of</strong><br />
Pediatrics, Vanderbilt <strong>University</strong> Medical Center, Nashville, TN, USA. 4 Department <strong>of</strong> Genetics, Harvard<br />
Medical School, Boston, MA<br />
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The epicardium plays an important role in coronary vessel formation. Identifying the genes and signaling<br />
pathways which regulate epicardial cell behavior during development may provide key insights not only into<br />
coronary vessel development but also epicardial-mediated cardiac repair in adults. Tgfbr3 -/- mice exhibit failed<br />
coronary vessel development associated with decreased epicardial cell invasion and proliferation, yet the exact<br />
mechanism is unknown. Tgfbr3 +/+ and Tgfbr3 -/- epicardial cells were incubated with ligands that drive invasion<br />
via TGFbR3, TGFb1 [250 pM], TGFb2 [250 pM], or BMP2 [5000 pM], to identify genes dysregulated after<br />
receptor loss. 72 hours after ligand addition cells were harvested for RNA sequencing analysis. 10 million<br />
reads identified ~14,000 significantly expressed genes per condition. Several genes were found to be 2-fold<br />
differentially expressed between Tgfbr3 +/+ and Tgfbr3 -/- cells (p
5.24 Dioxin Exposure Impairs Epicardium Development in the Zebrafish and Mouse<br />
Peter H<strong>of</strong>steen 1 , Jess Plavicki 1 , Richard E. Peterson 1 and Warren Heideman 1<br />
1 School <strong>of</strong> Pharmacy, <strong>University</strong> <strong>of</strong> Wisconsin, Madison, WI, USA<br />
The vertebrate primordial heart is comprised <strong>of</strong> the myocardium and endocardium. Further development <strong>of</strong> the<br />
heart relies heavily on the formation <strong>of</strong> the epicardium, which is the outer-most layer <strong>of</strong> the heart. The<br />
epicardium is derived from a transient extra-cardiac mesothelial organ called the proepicardium (PE). In<br />
zebrafish (Danio rerio), exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin, TCDD) at 24 hours post<br />
fertilization blocks formation <strong>of</strong> the epicardium. We hypothesized that failure to form the epicardium might be<br />
secondary to a TCDD-induced disruption <strong>of</strong> PE formation. To test this hypothesis, we scored for the presence or<br />
absence <strong>of</strong> the PE in zebrafish embryos exposed to TCDD. From our experiments it is clear that TCDD-exposed<br />
zebrafish fail to form a PE. Furthermore, in situ hybridization and immunohistochemistry revealed that<br />
transcription factor 21 (tcf21) expression was absent at the PE site following TCDD exposure. Cardiac<br />
malformations caused by TCDD exposure are not limited to zebrafish. Embryonic chick (Gallus gallus<br />
domesticus) and mouse (Mus musculus) hearts are also targets <strong>of</strong> TCDD toxicity; therefore, we broadened our<br />
investigation and hypothesized that TCDD causes PE or epicardial defects in these other vertebrates. TCDD<br />
exposure did not inhibit formation <strong>of</strong> the PE or epicardium in the chick and mouse. However, the TCDD-exposed<br />
mouse epicardium was distended from the underlying myocardium at E12. Supported by NIH Grant ES012716<br />
and UW Sea Grant.<br />
5.25 Rescue <strong>of</strong> Coronary Development by PDGF-BB and Sonic Hedgehog in FOG-2 Deficient Hearts<br />
Harold E. Olivey 1 , Alleda E. Flagg 2 and Eric C. Svensson 2<br />
1 Department <strong>of</strong> Biology, Indiana <strong>University</strong> Northwest, Gary, IN, 46408<br />
2 Department <strong>of</strong> Medicine, The <strong>University</strong> <strong>of</strong> <strong>Chicago</strong>, <strong>Chicago</strong>, IL, 60637<br />
Homozygous targeted mutation <strong>of</strong> the transcriptional co-repressor FOG-2 in mice results in mid-gestational<br />
embryonic death due to pr<strong>of</strong>ound structural abnormalities in the heart. Interestingly, FOG-2 deficient animals<br />
form an intact epicardium, but fail to elaborate a coronary vascular plexus by embryonic day (e) 12.5. In<br />
contrast, wild-type littermates have a well-developed coronary plexus by e12.5, suggesting that the loss <strong>of</strong> FOG-<br />
2 prevents coronary plexus formation. Taking a candidate gene approach, we identified two genes, PDGF-B and<br />
Sonic Hedgehog (Shh), that are downregulated in the hearts <strong>of</strong> FOG-2 deficient animals. We observe that<br />
PDGF-BB and Shh protein can each rescue coronary plexus formation in mutant hearts grown in vitro. Further<br />
we demonstrate that cyclopamine, a potent inhibitor <strong>of</strong> Shh signaling, prevents coronary plexus rescue by<br />
PDGF-BB, suggesting that PDGF-BB works upstream <strong>of</strong> Shh. Collectively our observations support a model<br />
where FOG-2 supports coronary vasculogenesis through upregulation <strong>of</strong> PDGF-BB and Shh expression. Our<br />
preliminary results suggest that FOG-2, PDGF-BB and Shh may act together in a pathway required for coronary<br />
vasculogenesis. Future experiments will attempt to define more precisely both the mechanism by which FOG-2<br />
regulates PDGF-BB expression and the other molecules involved in the FOG-2/PDGF-BB/Shh pathway.<br />
5.25 2,3,7,8-Tetrachlorodibenzo-p-dioxin Exposure Prevents Epicardium Formation in Zebrafish<br />
J.Plavicki, P. H<strong>of</strong>steen, R.E. Peterson and W. Heideman<br />
School <strong>of</strong> Pharmacy, <strong>University</strong> <strong>of</strong> Wisconsin, Madison, WI<br />
Embryonic exposure to the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) disrupts<br />
cardiac development in a wide range <strong>of</strong> vertebrate species including fish, birds and mammals. The temporal<br />
window <strong>of</strong> TCDD sensitivity in zebrafish embryos coincides with the formation <strong>of</strong> the epicardium. We therefore<br />
hypothesized that TCDD-induced heart failure in zebrafish results from disruptions in epicardial development.<br />
Using in situ hybridization, histology and fluorescence immunocytochemistry in conjunction with confocal<br />
microscopy, we have found that early embryonic TCDD exposure prevents the formation <strong>of</strong> the proepicardium<br />
(PE) and epicardium. Exposure to TCDD after PE formation also disrupts epicardial development. An early<br />
response to TCDD exposure is reduced cardiac output; therefore we investigated whether impaired myocardial<br />
function alone was sufficient to disrupt epicardial development. To prevent myocardial contraction, we injected<br />
silent heart (sih) morpholinos (MO) and scored for PE and epicardium formation. The PE was present in sih MO<br />
injected larvae, however the epicardium did not form. Next, we impaired myocardial contraction<br />
pharmacologically by applying the reversible myosin ATPase inhibitor, 2,3-Butanedione 2-monoxime (BDM).<br />
Application <strong>of</strong> BDM during PE specification and growth (24 to 72 hours post fertilization; hpf) did not prevent the<br />
formation <strong>of</strong> the PE or the epicardium. However, application <strong>of</strong> BDM during epicardial cell migration (72 to 120<br />
133
hpf and 96 to 120 hpf) stopped the further spreading <strong>of</strong> epicardial cells. Our results show that TCDD exposure<br />
prevents the formation <strong>of</strong> an essential layer <strong>of</strong> the vertebrate heart and that myocardial function is necessary for<br />
development <strong>of</strong> the epicardium in zebrafish.<br />
5.27 Heterozygous deletion <strong>of</strong> Wilms’ tumour-1 decreases fetal but not adult coronary artery<br />
development<br />
Carmen Leung, Xiangru Lu, Murong Liu, Qingping Feng<br />
Department <strong>of</strong> Physiology & Pharmacology, Collaborative Program in Developmental Biology, Lawson Health<br />
Research Institute, <strong>University</strong> <strong>of</strong> Western Ontario<br />
Congenital heart defects (CHD) are the most common human birth defect, occurring in approximately 5% <strong>of</strong><br />
newborns. As a major cause <strong>of</strong> morbidity and mortality, a further understanding <strong>of</strong> the mechanisms underlying<br />
CHD is crucial. During heart development, Wilms’ tumour-1 (Wt1) is expressed in epicardial cells and is critical<br />
for epicardium progenitor cell function, which gives rise to smooth muscle cells and fibroblasts that form<br />
coronary arteries. Wt1 -/- mice have severe cardiac defects and are embryonic lethal. Conversely, Wt1 +/- mice are<br />
viable and fertile but whether cardiac developmental defects occur due to a gene dosage effect remains elusive.<br />
We hypothesize that Wt1 +/- mice will exhibit decreased coronary artery formation during development. Postnatal<br />
day one (P1) hearts were immunostained for α-smooth muscle actin and 3-D reconstructions were generated to<br />
evaluate coronary artery volume and morphology. Wt1 +/- P1 mice exhibit significantly decreased coronary artery<br />
volume and coronary artery openings. There were no differences in heart size and total body weight. Wt1 +/-<br />
mice had significantly lower expression <strong>of</strong> HIF1α at P1. By P3, both HIF1α and VEGFa were significantly<br />
increased. By P5, no significant differences were found in HIF1α and VEGFa. In adult Wt1 +/- mice, no<br />
differences in coronary artery openings and capillary density were observed. We conclude that heterozygous<br />
deletion <strong>of</strong> Wt1 decreases coronary artery development in fetal but not adult mice. Our data suggests that<br />
compensatory mechanisms develop postnatal in the Wt1 +/- mice that lead to normal coronary artery formation in<br />
adulthood, potentially through the upregulation <strong>of</strong> HIF1α and VEGFa expression.<br />
5.28 p38 MAPK and Src kinase function in TGFβ2 mediated epicardial cell invasion and differentiation.<br />
Patrick B. Allison 1 , Derrick M. Broka 1 , Todd D. Camenisch 1,2,3,4<br />
1 <strong>University</strong> <strong>of</strong> Arizona, Dept. Pharmacology and Toxicology, Tucson, AZ, USA. 2 Southwest Environmental<br />
Health Sciences Center, <strong>University</strong> <strong>of</strong> Arizona, Tucson, AZ, USA. 3 Steele Children’s Research Center,<br />
<strong>University</strong> <strong>of</strong> Arizona, Tucson, AZ, USA. 4 Bio5 Institute, <strong>University</strong> <strong>of</strong> Arizona, Tucson, AZ, USA<br />
The epicardium undergoes proliferation, migration and differentiation into several cardiac cell types including<br />
smooth muscle that will contribute to the coronary vessels. Although the requirement for TGFβ family <strong>of</strong> ligands<br />
and receptors for epicardial epithelial to mesenchymal transition (EMT) have been recently characterized, the<br />
significance <strong>of</strong> downstream TGFβ signaling effectors have yet to be addressed. Both p38 MAP kinase and Src<br />
Kinase have recently been implicated in TGFβ signal transduction, but their potential roles in epicardial cell<br />
biology are unclear. Utilizing a primary murine epicardial cell model, we addressed the role <strong>of</strong> p38 and Src in<br />
TGFβ2 stimulated invasion and differentiation. Epicardial cells were treated with small molecule inhibitors and<br />
siRNA to p38 and Src, subsequently stimulated with TGFβ2, and allowed to differentiate. We demonstrate<br />
blocking p38 and Src activity results in inhibited TGFβ2 induction <strong>of</strong> pro-EMT genes such as CD44 and<br />
Hyaluronan Synthase 2, and block down-regulation <strong>of</strong> the TGFβ type III receptor. We show blocking p38 and<br />
Src leads to reduced TGFβ2 induction <strong>of</strong> epicardial Hyaluronan production. We evaluated the importance <strong>of</strong><br />
p38 and Src kinase in TGFβ2 stimulated smooth muscle differentiation using a β-galactosidase reporter for<br />
smooth muscle actin 22α. Finally, blocking p38 and Src attenuates TGFβ2 induced invasion using a trans-well<br />
invasion assay and EMT in collagen gel cultures. Collectively, our data suggests p38 and Src kinase function in<br />
TGFβ2-mediated epicardial cell differentiation and invasion.<br />
5.29 Somatopleural origin <strong>of</strong> a proepicardial cell population in the avian embryo<br />
Jan Schlueter and Thomas Brand<br />
Harefield Heart Science Centre, National Heart and Lung Institute, Imperial College London, United Kingdom<br />
The proepicardium (PE) develops at the venous pole <strong>of</strong> the tubular heart and in many vertebrates including the<br />
avian embryo PE development is asymmetric. Only the mesothelial cell cluster on the right side generates a<br />
tissue bridge via which proepicardial cells colonize the heart to form the epicardium. PE lateralization is under<br />
the control <strong>of</strong> a signaling pathway involving FGF8 and SNAI1 (Schlueter and Brand (2009) PNAS 106:7485-90).<br />
Expression analysis <strong>of</strong> TWIST1, which represents a potential target <strong>of</strong> the FGF8/SNAI1 pathway, revealed<br />
134
transient expression in the right somatopleura and subsequently in the right sinus horn. Forced expression <strong>of</strong><br />
SNAI1 on the left side caused ectopic TWIST1 expression suggesting that SNAI1 is upstream <strong>of</strong> TWIST1. The<br />
expression pattern <strong>of</strong> TWIST1 indicated that some PE cells might have a somatopleural origin. We performed<br />
cell fate experiments and observed a contribution <strong>of</strong> somatopleural cells to the sinus venosus and the PE. After<br />
forced expression <strong>of</strong> TWIST1 on the left side, somatopleural cells invaded the sinus venosus and the heart,<br />
suggesting that TWIST1 is able to enhance cell migration into the venous pole <strong>of</strong> the heart. Suppression <strong>of</strong><br />
TWIST1 expression by shRNAi on the right side resulted in impaired proepicardial development and<br />
suppression <strong>of</strong> WT1 expression. Currently we are testing whether the loss <strong>of</strong> TWIST1 also leads to alterations in<br />
the cellular differentiation potential <strong>of</strong> proepicardial explants. These data suggest that the PE in the avian<br />
embryo is made up <strong>of</strong> distinct cell populations, which differ in their embryological origin.<br />
5.30 Partial absence <strong>of</strong> pleuropericardial membranes in Tbx18- and Wt1-deficient mice<br />
Julia Norden 1 , Thomas Grieskamp 1 , Antoon Moorman 2 , Vincent Christ<strong>of</strong>fels 2 , and Andreas Kispert 1<br />
1<br />
Institut für Molekularbiologie, Medizinische Hochschule Hannover, 30625 Hannover, Germany.<br />
2<br />
Heart Failure Research Center, Academic Medical Center, <strong>University</strong> <strong>of</strong> Amsterdam, 1105 AZ Amsterdam, The<br />
Netherlands<br />
The pleuropericardial membranes are fibro-serous walls that separate the pericardial and pleural cavities and<br />
anchor the heart inside the mediastinum. Partial or complete absence <strong>of</strong> pleuropericardial membranes is a rare<br />
human disease, the etiology <strong>of</strong> which is poorly understood. As an attempt to better understand these defects,<br />
we wished to analyze the cellular and molecular mechanisms directing the separation <strong>of</strong> pericardial and pleural<br />
cavities by pleuropericardial membranes in the mouse. We found by histological analyses that both in Tbx18-<br />
and Wt1-deficient mice the pleural and pericardial cavities communicate due to a partial absence <strong>of</strong> the<br />
pleuropericardial membranes in the hilus region, and we trace these defects to a persistence <strong>of</strong> the embryonic<br />
communication between these cavities, the pericardioperitoneal canals. Furthermore, we identify mesenchymal<br />
ridges in the sinus venosus region that tether the growing pleuropericardial membranes to the hilus <strong>of</strong> the lung,<br />
and thus, close the pericardioperitoneal canals. In Tbx18-deficient embryos these mesenchymal ridges are not<br />
established, whereas in Wt1-deficient embryos the final fusion process between these tissues and the body wall<br />
does not occur. We suggest that this fusion is an active rather than a passive process, and discuss the<br />
interrelation between closure <strong>of</strong> the pericardioperitoneal canals, lateral release <strong>of</strong> the pleuropericardial<br />
membranes from the lateral body wall, and sinus horn development.<br />
Section 6: Early Heart Formation and Progenitors<br />
S6.1 Quantitative analysis <strong>of</strong> polarity in 3D reveals local cell coordination in the embryonic mouse heart<br />
Jean-François Le Garrec 1 , Chiara Ragni 1 , Sorin Pop 2 , Alexandre Dufour 2 , Jean-Christophe Olivo-Marin 2 ,<br />
Margaret Buckingham 1 and Sigolène Meilhac 1<br />
1 2<br />
Dept <strong>of</strong> Developmental Biology, CNRS URA 2578, Dept <strong>of</strong> Cell Biology and Infection, CNRS URA 2582,<br />
Institut Pasteur, 75015 Paris, France<br />
Several mutations in genes encoding members <strong>of</strong> the Planar Cell Polarity pathways impair heart<br />
morphogenesis. However, no asymmetric localization <strong>of</strong> such proteins has been reported in myocardial cells<br />
and it has remained unclear how the embryonic myocardium is polarized. We have now identified cell polarity<br />
markers in the mouse and aim to quantify the degree <strong>of</strong> coordination between myocardial cells in the embryonic<br />
heart. Classically, anisotropies which underlie organ morphogenesis have been quantified as a 2D problem,<br />
taking advantage <strong>of</strong> a reference axis. However, this is not applicable to the looped heart tube, which has a<br />
complex geometry. We have designed a 3D image processing framework, to map the regions in which cell<br />
polarities are significantly aligned. This novel procedure integrates multidisciplinary tools, including image<br />
segmentation, statistical analyses, axial clustering and correlation analysis. The result is a sensitive and<br />
unbiased assessment <strong>of</strong> the significant alignment <strong>of</strong> cell orientations in 3D, compared to a random axial<br />
distribution. We show that the axes <strong>of</strong> cell polarity, defined as the centrosome-nucleus axes, are frequently<br />
biased in a plane parallel to the outer surface <strong>of</strong> the heart, with a minor transmural component. This reflects the<br />
expansion <strong>of</strong> the cardiac chambers which precedes transmural thickening. Our study reveals that ventricular<br />
cells locally coordinate their axes over 100µm. This is in keeping with the growth <strong>of</strong> the myocardium, that we<br />
had shown by clonal analysis to be regionally oriented.<br />
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S6.2 Clonally dominant cardiomyocytes direct heart morphogenesis<br />
Vikas Gupta and Kenneth D. Poss<br />
Department <strong>of</strong> Cell Biology and Howard Hughes Medical Institute, Duke <strong>University</strong> Medical Center, Durham,<br />
North Carolina 27710, USA<br />
As vertebrate embryos develop into adulthood, their organs dramatically increase in size and change tissue<br />
architecture. Here, we used a multicolor clonal analysis to define the contributions <strong>of</strong> many individual<br />
cardiomyocytes as the zebrafish heart undergoes morphogenesis from a primitive embryonic structure into its<br />
complex adult form. We find that the single cardiomyocyte-thick wall <strong>of</strong> the juvenile ventricle forms by lateral<br />
expansion <strong>of</strong> several dozen cardiomyocytes into muscle patches <strong>of</strong> variable sizes and shapes. As juveniles<br />
mature into adults, this structure becomes fully enveloped by a new lineage <strong>of</strong> cortical muscle. Adult cortical<br />
muscle originates from a small number (~8) <strong>of</strong> cardiomyocytes that display clonal dominance reminiscent <strong>of</strong><br />
stem cell populations. Cortical cardiomyocytes initially emerge from internal my<strong>of</strong>ibers that in rare events<br />
breach the juvenile ventricular wall and expand over the surface. Our study illuminates dynamic proliferative<br />
behaviors that generate adult cardiac structure, revealing clonal dominance as a key mechanism that shapes a<br />
vertebrate organ.<br />
S6.3 Gα 13 is required for S1pr2-mediated myocardial migration by regulating endoderm morphogenesis<br />
Ding Ye 1 , Songhai Chen 2 , Fang Lin 1 *<br />
1 Department <strong>of</strong> Anatomy and Cell Biology, 2 Department <strong>of</strong> Pharmacology, Carver College <strong>of</strong> Medicine,<br />
<strong>University</strong> <strong>of</strong> Iowa, 1-400 Bowen Science Building, 51 Newton Road, Iowa city, IA 52242-1109, USA<br />
During vertebrate development, bilateral populations <strong>of</strong> myocardial precursors migrate towards the midline to<br />
form the primitive heart tube, and this requires both their intrinsic properties and the appropriate environment,<br />
including endoderm. Disruption <strong>of</strong> myocardial migration leads to the formation <strong>of</strong> two bilaterally located hearts, a<br />
condition known as cardiac bifida. In zebrafish, signaling mediated by sphingosine-1-phosphate (S1P) and its<br />
cognate G protein-coupled receptor (S1pr2/Mil) controls myocardial migration. However, the underlying<br />
downstream mechanism remains poorly understood. Here we show that depletion <strong>of</strong> specifically the G protein<br />
is<strong>of</strong>orm Gα13, results in cardia bifida and tail blistering, defects reminiscent <strong>of</strong> those observed in embryos<br />
deficient for S1P signaling. Our genetic studies indicate that Gα13 acts downstream <strong>of</strong> S1pr2 to regulate<br />
myocardial migration through a RhoGEF/Rho-dependent pathway. Intriguingly, both cardiomyocytes and<br />
endoderm express S1pr2 and Gα13, and cardiac-specific expression <strong>of</strong> Gα13 fails to rescue cardia bifida in the<br />
context <strong>of</strong> global Gα13 inhibition. Furthermore, we found that disruption <strong>of</strong> any component <strong>of</strong> the<br />
S1pr2/Gα13/RhoGEF signaling pathway led to defects in endoderm morphogenesis that were correlated to<br />
cardia bifida, and that defects in both endoderm and myocardial migration resulting from Gα13 depletion were<br />
rescued coincidently. Overall, our findings represent the first evidence that S1pr2 controls myocardial migration<br />
through a Gα13/RhoGEF-dependent pathway, and that it does so by regulating endoderm morphogenesis.<br />
Currently, we are investigating the mechanisms by which S1pr2/Gα13 signaling controls endoderm<br />
morphogenesis.<br />
S6.4 A Cdc42 associated genetic network directs heart lumen formation and morphogenesis in<br />
Drosophila<br />
Georg Vogler 1 , Jiandong Liu 2 and Rolf Bodmer 1<br />
1 Development and Aging Program, Sanford-Burnham Medical Research Institute, La Jolla, CA<br />
2 Department <strong>of</strong> Biochemistry and Biophysics, <strong>University</strong> <strong>of</strong> California, San Francisco, CA.<br />
The Drosophila embryonic heart is a key model system for understanding heart specification. Our previous<br />
studies indicate that heart morphogenesis requires Slit/Robo signaling, a function conserved in vertebrates. The<br />
mechanisms by which these and other signals control heart formation are still unknown. Due to its role in<br />
membrane dynamics, we investigated the role <strong>of</strong> the small GTPase Cdc42 during Drosophila heart development<br />
and found it to be required for cardiac cell alignment and heart tube formation. Mutant or constitutively active<br />
Cdc42 in the developing heart causes improper cardioblast alignment and formation <strong>of</strong> multiple lumina,<br />
suggesting that Cdc42 is required during discrete steps <strong>of</strong> cardiogenesis. Cell polarity and filopodia dynamics<br />
are unaffected by loss <strong>of</strong> Cdc42, therefore Cdc42 might have a different role during heart morphogenesis. To<br />
understand the regulation <strong>of</strong> Cdc42 and to identify new genetic interactors, we performed a genetic screen for<br />
modifiers <strong>of</strong> Cdc42. We identified the tyrosine kinase Abelson (Abl), and the non-muscle myosin-II zipper to<br />
strongly interact with Cdc42. Abl itself shows a requirement for coordinated heart tube assembly, and Zipper<br />
exhibits a dynamic localization pattern during cardiogenesis, which depends on Cdc42 function, but is<br />
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independent <strong>of</strong> Slit/Robo. Activation <strong>of</strong> the formin-like protein Diaphanous (Dia) produced defects similar to<br />
activated Cdc42, indicating that control <strong>of</strong> cell shape changes is a key regulatory step during heart<br />
morphogenesis. Our data suggest a novel mechanism <strong>of</strong> cardiac morphogenesis involving Abl, Cdc42, Dia and<br />
Zip acting in a common pathway during cardiac cell shape changes and orchestrated heart lumen formation.<br />
S6.5 Identification and characterization <strong>of</strong> a multipotent cardiac precursor<br />
W. Patrick Devine, Josh D. Wythe, Benoit G. Bruneau<br />
Gladstone Institute <strong>of</strong> Cardiovascular Disease, J. David Gladstone Institute<br />
San Francisco, CA 94158<br />
Construction <strong>of</strong> the vertebrate heart is a complex process involving the regulated contribution <strong>of</strong> cardiac<br />
progenitor cells in a discrete spatial and temporal order. The precision required for this process is highlighted by<br />
the frequency <strong>of</strong> congenital heart defects. Understanding the identity and regulation <strong>of</strong> these progenitors is<br />
critical to understanding the origins <strong>of</strong> congenital heart defects and has the potential to lead to novel cell-based<br />
regenerative therapies for heart disease. Previous lineage tracing studies have predicted the existence <strong>of</strong> an<br />
early, multi-potent cardiovascular progenitor, but the identity <strong>of</strong> this progenitor has remained undefined. The<br />
transcription factor Mesp1 has been postulated to mark early cardiac progenitors, however our lineage tracing<br />
results suggest that Mesp1 labels a much broader domain <strong>of</strong> cardiac and non-cardiac mesoderm than initially<br />
reported. Baf60c/Smarcd3, a subunit <strong>of</strong> the BAF chromatin-remodeling complex, is expressed specifically in the<br />
heart and somites in the early mouse embryo. We have identified an early domain <strong>of</strong> Smarcd3 expression, prior<br />
to formation <strong>of</strong> the cardiac crescent in a region <strong>of</strong> the late gastrula stage mouse embryo predicted to contain a<br />
cardiovascular progenitor population. Temporally-regulated lineage tracing <strong>of</strong> this population specifically labels<br />
both first and second heart field derived structures, including the endocardium, myocardium, and epicardium,<br />
and a population <strong>of</strong> cells within the anterior forelimb. Ongoing work will describe the precise temporal and<br />
spatial localization <strong>of</strong> Smarcd3 relative to other markers <strong>of</strong> early cardiogenesis (Tbx5, Isl1, and Nkx2-5) as well<br />
as the clonal relationship among these early Smarcd3-expressing cells.<br />
S6.6 Distinct origin and commitment <strong>of</strong> HCN4+ First Heart Field cells towards cardiomyogenic cell<br />
lineages<br />
Daniela Später 1,2 , Monika Abramczuk 1,2,3 , Jon Clarke 1,2 , Kristina Buac 1,2 , Makoto Sahara 1,2 , Andreas Ludwig 4 ,<br />
Kenneth R. Chien 1,2<br />
1 Cardiovascular Research Center, Massachusetts General Hospital, Boston, USA. 2 Harvard Stem Cell<br />
Institute, Department <strong>of</strong> Stem Cell and Regenerative Biology, Cambridge, USA. 3 Institute <strong>of</strong> Molecular<br />
Biotechnology <strong>of</strong> the Austrian Academy <strong>of</strong> Sciences (IMBA), Vienna, Austria. 4 Institut für Experimentelle und<br />
Klinische Pharmakologie und Toxikologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Germany<br />
Most <strong>of</strong> the mammalian heart is formed from two anatomically and molecularly distinct groups <strong>of</strong> cells <strong>of</strong><br />
mesodermal origin termed the first and second heart fields (FHF and SHF). Whereas the SHF gives rise to the<br />
right ventricle, parts <strong>of</strong> the atria, and proximal region <strong>of</strong> the outflow-tract, the FHF gives rise to the left ventricle<br />
and remaining parts <strong>of</strong> the atria. Multipotent progenitors <strong>of</strong> the SHF are marked preferentially by Islet-1<br />
expression, while a marker exclusive to FHF progenitors has not been identified. Here, we present Hcn4, a gene<br />
encoding the hyperpolarization-activated cyclic nucleotide-gated channel 4, as a new FHF marker expressed as<br />
early as pre-crescent stage. In-situ hybridization analysis revealed a dynamic expression <strong>of</strong> Hcn4 in the<br />
developing heart, with its strongest expression in the cardiac crescent and down-regulation by E9.5. HCN4-<br />
CreErt2 lineage tracing experiments confirm that the progeny <strong>of</strong> these cells give rise to FHF-derived structures<br />
<strong>of</strong> the heart. Surprisingly, these cells contribute primarily to cardiomyogenic cell lineages, suggesting that Hcn4+<br />
FHF precursor cells are committed cardiomyogenic progenitors from their earliest detectable stage. Single cell<br />
clonal analysis using Hcn4+ FHF cells from both mouse embryos and differentiated mouse embryonic stem cells<br />
support this finding. Our data suggests a model <strong>of</strong> cardiogenesis, where the primary purpose <strong>of</strong> the FHF is to<br />
generate cardiac muscle to support the contractile activity <strong>of</strong> the primitive heart tube, whereas the contribution <strong>of</strong><br />
SHF-derived progenitors to the heart is more diverse, leading to cardiomyocytes, smooth muscle and<br />
endothelial cells.<br />
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6.7 Dynamic Mesp1 regulation directs cardiac myocyte formation in embryonic stem cells<br />
Yu Liu 1 , Xueping Xu 2 , Austin Cooney 2 , Robert Schwartz 1<br />
Department <strong>of</strong> Biology and Biochemistry, <strong>University</strong> <strong>of</strong> Houston<br />
Department <strong>of</strong> Molecular and Cellular Biology, Baylor College <strong>of</strong> Medicine<br />
Mesp1 sits on the top <strong>of</strong> the hierarchy <strong>of</strong> cardiac gene regulation network. It emerges at the onset <strong>of</strong> mesoderm<br />
formation and disappears at the cardiac crescent. The extracellular cue that triggers Mesp1, and how the<br />
Mesp1-expressing cells differentiate into cardiovascular cell lineages are largely unknown. In this study, we<br />
have crossed the Mesp1 Cre/+ and Rosa EYFP/EYFP mouse strains, and established an ES cell line with the genotype<br />
Mesp1 Cre/+ /Rosa EYFP/+. By isolating EYFP (+) cells on day 4 <strong>of</strong> an ES differentiation protocol, we characterized<br />
the early cardiac progenitors. These cardiac progenitors enriched cardiac mesoderm markers (Flk1, PDGFRa,<br />
dHand), cardiac transcription factors (Nkx2-5, Tbx5, Mef2c), and excluded pluripotent markers (Oct4, Sox2,<br />
Nanog) and nascent mesoderm markers (T, Fgf8). Among a group <strong>of</strong> growth factors, BMP2/4 greatly induced<br />
the prevalence <strong>of</strong> EYFP (+) cells, while Wnt3a and Activin had only marginal effects. EYFP(+) cells represented<br />
a sub-population <strong>of</strong> the Flk1(+)/PDGFRa(+) cells, which were previously demonstrated to differentiate into<br />
cardiac lineages. Surprisingly, short period <strong>of</strong> BMP4 treatment (day 0-2) led to the induction <strong>of</strong> EYFP(+) cells<br />
and subsequent cardiac myocyte formation, whereas extended BMP4 (day 0-4) led to even more EYFP (+) cells<br />
but without subsequent cardiac myocyte formation. By introducing a pCAGG-loxP-bgeo-polyA-loxP-Mesp1<br />
cassette into this ES cell line, we achieved extended Mesp1 expression in EYFP(+) cells beyond the point that<br />
endogenous Mesp1 disappears, and proved that extended Mesp1 expression impaired cardiac myocyte<br />
formation. In summary, we have established an ES cell line that allows us to trace the fate <strong>of</strong> Mesp1-expressing<br />
cardiac progenitors. Using this line as the tool, we demonstrate that dynamic Mesp1 regulation directs cardiac<br />
myocyte formation in ES cells.<br />
6.8 Human amniocytes contain subpopulations <strong>of</strong> stem cells that have a repressed cardiogenic status<br />
Colin T. Maguire, Bradley Demarest, Jennifer Akiona, Jonathon Hill, James Palmer, *Arthur R. Brothman, H.<br />
Joseph Yost, Maureen L. Condic<br />
Departments <strong>of</strong> Neurobiology and Anatomy and *Pediatrics and ARUP Laboratories, <strong>University</strong> <strong>of</strong> Utah<br />
Molecular Medicine Program, Eccles Institute <strong>of</strong> Human Genetics, Building 533, Room 3160, 15 North 2030<br />
East, Salt Lake City, Utah 84112-5330, USA<br />
Amniocytes are an intriguing candidate source <strong>of</strong> stem cells for autologous repair <strong>of</strong> congenital heart defects<br />
(CHD) in humans. Despite mesodermal fate poising, amniocytes are resistant to differentiating into fully<br />
functional cardiomyocytes. Here, we provide evidence that cultured amniocytes are a heterogeneous<br />
subpopulation <strong>of</strong> stem cells expressing key pluripotency and self-renewal markers. Interestingly, qPCR analysis<br />
<strong>of</strong> 17 independent amniocyte isolates also detected the expression <strong>of</strong> the core cardiogenic factors Mef2c, Tbx5,<br />
and Nkx2-5, as well as the cardiac chromatin regulator Baf60c (Smarcd3). Gata4 is not expressed, but some<br />
individual isolates show signs <strong>of</strong> a low level <strong>of</strong> gene leakiness. However, Tbx5 and the early-to-mid cardiac<br />
markers Mesp1, Isl1, and Gata6 were consistently detectable by both qPCR and immunostaining. Stimulating<br />
amniocytes with signals known to promote differentiation <strong>of</strong> cardiac lineages failed to upregulate mRNA<br />
transcript or protein levels <strong>of</strong> core cardiogenic factors or generate cells that stain positive for cardiac troponin T<br />
(CTNT) and MF20 markers. To more precisely define the cardiogenic status <strong>of</strong> amniocytes, we performed next<br />
generation RNA-seq analysis on amniocytes. Genome-wide expression pr<strong>of</strong>iling reveals a paradoxical<br />
molecular signature <strong>of</strong> cardiac markers, indicating that the developmental status <strong>of</strong> amniocytes as true cardiac<br />
progenitors is incomplete, yet uniquely poised. Amniocytes strongly express multiple ES cell and mesodermal<br />
repressors that may prevent cells from progressing further down a cardiogenic lineage. These results indicate<br />
that despite an incomplete cardiogenic phenotype, direct transdifferentiation and gene knockdown approaches<br />
may allow us to overcome these barriers and drive them to a mature cardiac cell fate. Supported in part by<br />
Primary Children’s Medical Foundation, T32HL007576 Cardiovascular Training Grant, and U01HL098179<br />
(NHLBI Bench-to-Bassinet program)<br />
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6.9 The Homeobox Transcription Factor, Irx4, Identifies a Mutipotent, Ventricular-specific Cardiac<br />
Progenitor<br />
Daryl Nelson 1 , Joshua Trzebiatowski 1 , Erin Willing 1 , Pearl Hsu 2 , Kevin Schoen 1 , Kevin Liberko 1 , Matthew<br />
Butzler 1 , Pat Powers 2 , Manorama John 2 , Timothy Kamp 1 , Gary Lyons 1<br />
1 School <strong>of</strong> Medicine and Public Health, <strong>University</strong> <strong>of</strong> Wisconsin-Madison<br />
2 <strong>University</strong> <strong>of</strong> Wisconsin Biotechnology Center<br />
Cardiac progenitors have been presented as potential cell therapeutics, due to their cardiogenic potency.<br />
Iroquois homeobox protein 4 (Irx4) is the earliest known marker <strong>of</strong> ventricular myocardium differentiation, and<br />
the transcription factor is restricted to the ventricles throughout embryogenesis and into adulthood. Our goal is<br />
to identify a multipotent, ventricular-specific cardiac progenitor. We have targeted the 3’ end <strong>of</strong> the Irx4 locus<br />
using a recombineering approach to insert fluorescence (mCherry), bioluminescence (luciferase), and antibiotic<br />
resistance (hph) cassettes. Six mESC clones were properly targeted Irx4 luc-mCh-hph/wt<br />
ES cells. RT-PCR, western<br />
blot analysis, and immun<strong>of</strong>luorescence assays demonstrated the functional integration <strong>of</strong> the reporters during<br />
embryoid body (EB) differentiation. Following 4 days <strong>of</strong> differentiation <strong>of</strong> the Irx4 luc-mCh-hph/wt cells in EBs,<br />
selection with hygromycin was carried out for 2 days, and day 6 cells were plated onto STO cell feeder layers for<br />
expansion. Selected Irx4 + cells are proliferative, expressing the cell cycle antigen, Ki67, and could be<br />
passaged more than 12 times. The Irx4 + cells express cKit, Flk1, and CXCR4 on the cell surface. Selected<br />
Irx4 + cells demonstrate cardiovascular potency when re-aggregated and differentiated in hanging drops resulted<br />
in ventricular myocyte-enriched cell preparations (65±3.7% cTnT + ; 67±2.6% Myl2 + , 22±3.1% SmMHC + , and<br />
6±2% CD31 + cells. The selected Irx4 + population represents the first ventricular-specific multipotent progenitor<br />
population identified and holds promise for generating all cardiovascular lineages necessary to reconstitute<br />
infarcted myocardium.<br />
6.10 Modeling the vascular phenotype <strong>of</strong> Williams-Beuren syndrome using induced pluripotent stem<br />
cells<br />
Caroline Kinnear, Wing Y. Chang, Shahryar Khattak, Karen Kennedy, Naila Mahmut, Tadeo Thompson,<br />
Aleksander Hinek, Gordon Keller, William L. Stanford, James Ellis, Seema Mital.<br />
Hosp for Sick Children, Toronto, ON, Canada<br />
Elastin is essential for arterial morphogenesis. Elastin haploinsufficiency in Williams-Beuren syndrome (WBS)<br />
leads to increased vascular smooth muscle cell (SMC) proliferation and vascular stenoses. We investigated the<br />
role <strong>of</strong> elastin in proliferation and differentiation <strong>of</strong> SMCs derived from human embryonic stem cells (hESCs),<br />
and WBS patient-derived induced pluripotent stem cells (hiPSCs). Human iPSCs were reprogrammed from skin<br />
fibroblasts from a WBS patient and BJ healthy control using 4-factor retrovirus reprogramming. hESCs and<br />
hiPSCs underwent directed differentiation to generate smooth muscle cells (SMCs). Differentiated cells were<br />
treated with a peptide or an anti-proliferative drug for 6 days. mRNA and protein expression <strong>of</strong> elastin, smooth<br />
muscle α-actin (SMA), Ki67 (proliferation marker) was assessed by high-content imaging, qRT-PCR, and flow<br />
cytometry. Vascular tube formation was assessed by 3D matrigel assays. Treatment <strong>of</strong> hESC-derived SMCs<br />
increased elastin expression, increased SMA+ cells, and reduced SMC proliferation. The reprogrammed<br />
fibroblasts expressed pluripotency genes and differentiated into all germ layers. SMC differentiation was<br />
confirmed by SMA, SM22a, myocardin, smoothelin expression and by contractile response to carbachol. When<br />
compared to BJ SMCs, WBS SMCs showed lower expression <strong>of</strong> elastin protein and mRNA which increased with<br />
treatment. WBS cells showed impaired SMC differentiation, increased SMC proliferation, and impaired vascular<br />
tube formation compared to BJ cells. Treatment with an elastin ligand and a candidate drug reduced SMC<br />
proliferation, enhanced SMC differentiation, and enhanced tube formation. In conclusion, WBS iPSCs<br />
demonstrated increased SMC proliferation and impaired SMC differentiation which was partially ameliorated<br />
with treatment. Our study provides an in vitro platform for modeling vascular disorders and future drug screens.<br />
6.11 Galnt11 is a Novel Glycosylation Factor that Interacts with Notch to Affect Left-Right Development<br />
and Heart Formation<br />
Marko T. Boskovski, 1 Svetlana Makova, 1 Martina Brueckner 1 and Mustafa Khokha 1<br />
1 Yale School <strong>of</strong> Medicine, Departments <strong>of</strong> Pediatrics and Genetics<br />
Heterotaxy is a disease <strong>of</strong> abnormal left-right (LR) body patterning associated with congenital heart disease that<br />
has a significantly increased prevalence in patients with ciliopathies. Through copy number variant analysis <strong>of</strong><br />
Heterotaxy patients, Galnt11 was identified as a glycosylation factor important in human LR development.<br />
Morpholino (MO) knockdown and mRNA overexpression <strong>of</strong> Galnt11 in X. tropicalis results in abnormal heart<br />
139
looping, and the splice-site MO phenotype can be rescued with GALNT11 mRNA. Sided MO injections at the 2cell<br />
stage revealed that Galnt11 is only required on the left side for proper heart looping. Knockdown and<br />
overexpression also results in abnormal Coco and PitX2 (early LR markers) expression patterns via in situ<br />
hybridization. Galnt11 is expressed in the X. tropicalis gastrocoel ro<strong>of</strong> plate (GRP) and kidneys, and in mouse<br />
Galnt11 protein localizes to the crown cells surrounding the pit cells <strong>of</strong> the node. This points to a ciliary role <strong>of</strong><br />
Galnt11, which we investigated on the X. tropicalis ciliated epidermis. Galnt11 knockdown yields an increase,<br />
while Galnt11 overexpression yields a decrease in cilia clump density, which is a known Notch phenotype. To<br />
further investigate the relationship between Galnt11 and Notch, we were able to rescue abnormal PitX2<br />
expression in Galnt11 morphants by injecting mRNA <strong>of</strong> two Notch components downstream <strong>of</strong> Notch receptor,<br />
Notch-ICD and Su(H)-Ank, but not by injecting Delta, a Notch ligand. These results indicate that Galnt11 is a<br />
glycosylation factor that interacts with Notch, possibly at the level <strong>of</strong> Notch receptor, to affect proper LR<br />
development and heart formation.<br />
6.12 Zic3 is required in the migrating primitive streak for node morphogenesis and left -right patterning<br />
Mardi J. Sutherland, Shuyun Wang, Malgorzata E. Quinn, Allison Haaning, Stephanie M. Ware<br />
Cincinnati Children’s Hospital Medical Center, Division <strong>of</strong> Molecular Cardiovascular Biology and <strong>University</strong> <strong>of</strong><br />
Cincinnati College <strong>of</strong> Medicine<br />
In humans, loss <strong>of</strong> function mutations in ZIC3 cause X-linked heterotaxy, a disorder characterized by abnormal<br />
left-right asymmetry <strong>of</strong> organs and frequent cardiovascular malformations. Zic3 is expressed ubiquitously during<br />
critical stages for left-right patterning but its later expression in the developing heart remains controversial and<br />
the molecular mechanism(s) by which it causes heterotaxy are unknown. Zic3 null mice recapitulate the human<br />
heterotaxy phenotype but also have early gastrulation defects, complicating an assessment <strong>of</strong> the role <strong>of</strong> Zic3 in<br />
cardiac development. To define the temporal and spatial requirement for Zic3 in left-right patterning, we<br />
generated conditional Zic3 mice and Zic3-LacZ-BAC reporter mice. The latter provide compelling data indicating<br />
that Zic3 is expressed in the mouse node. We hypothesized that Zic3 expression in node cells is required for<br />
proper left-right asymmetry. To address this question, Zic3 was deleted in each cell type <strong>of</strong> the node using a<br />
conditional loss <strong>of</strong> function approach. Surprisingly, Zic3 deletion in the node results in viable, phenotypically<br />
normal mice. However, immunohistochemistry and scanning electron microscopy indicate abnormal node<br />
morphology in Zic3 null mice. We observed similar node dysplasia when Zic3 was deleted from the migrating<br />
mesoderm and primitive streak at E7.5. At later stages, heart looping defects were observed. Interestingly, mice<br />
were viable when Zic3 was deleted from the heart compartment and heart progenitors. These results contrast<br />
with the accepted paradigm that left-right asymmetry is initiated at the node in mice and implicate Zic3 in earlier<br />
signaling events affecting node formation and subsequent node function. This work was funded by RO1<br />
HL088639 from the National Institutes <strong>of</strong> Health (S.M.W.) and NIH T32 HL007752-16 Pulmonary and<br />
Cardiovascular Developmental Training Grant (J.A.W.)<br />
6.13 The Vacuolar ATPase Accessory Protein Atp6ap1 Controls Heart Laterality in Zebrafish<br />
Jason Gokey and Jeffrey D. Amack<br />
Department <strong>of</strong> Cell and Developmental Biology, State <strong>University</strong> <strong>of</strong> New York, Upstate Medical <strong>University</strong>,<br />
Syracuse, NY, USA<br />
Establishing left-Right (LR) asymmetry, or laterality, <strong>of</strong> the primitive heart tube is critical for subsequent cardiac<br />
form and function. Small molecule screens have identified a role for the Vacuolar Type ATPase (V-ATPase) in<br />
establishing heart laterality, but the underlying mechanisms remain unclear. The V-ATPase is a multi-subunit<br />
proton pump in membranes that maintain organelle and cellular pH by pumping protons into the organelle lumen<br />
and out <strong>of</strong> the cellular cytoplasm. In the zebrafish embryo, the V-ATPase accessory protein Atp6ap1 is<br />
prominently expressed in a transient organ called Kupffer’s vesicle (KV). Motile cilia that project into the KV<br />
lumen generate an asymmetric fluid flow that is required for normal cardiac laterality. Depletion <strong>of</strong> Atp6ap1 using<br />
antisense morpholinos caused heart LR defects and disrupted the formation <strong>of</strong> KV. Atp6ap1 knockdown<br />
significantly decreased the length and number <strong>of</strong> KV cilia and reduced KV lumen size. These KV defects were<br />
rescu ed by ectopic Atp6ap1 expression. Interestingly, over-expression <strong>of</strong> Atp6ap1 also disrupted KV<br />
development and heart laterality, suggesting tight control <strong>of</strong> V-ATPase activity is essential. Consistent with this<br />
idea, interfering with V-ATPase function via morpholino knockdowns <strong>of</strong> V-ATPase subunits or treatments with<br />
the small molecule V-ATPase inhibitor concanamycin resulted in similar KV and cardiac LR defects. These<br />
results indicate V-ATPase function regulates KV formation, a critical step in establishing laterality <strong>of</strong> the heart.<br />
Next, we will elucidate mechanisms by which the V-ATPase regulates KV development and heart LR asymmetry<br />
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y examining potential connections between V-ATPase activity and the Notch, FGF and Wnt signaling<br />
pathways.<br />
6.14 A potential role for IGFBP5 in left-right cardiac specification<br />
Andrew Robson, Martina Brueckner and Mustafa Khokha<br />
Department <strong>of</strong> Pediatrics, Yale <strong>University</strong> Medical School, New Haven, Connecticut<br />
Heterotaxy (Htx) is a condition where the normal left-right (LR) asymmetry is not properly established. Cilia<br />
present at the LR organiser establish the LR axis. Using a genome-wide analysis <strong>of</strong> copy number variations<br />
(CNVs) in Htx patients we have identified a genomic deletion in the Insulin Growth Factor Binding Protein<br />
(IGFBP5) a novel gene previously unrecognised in LR patterning. We have used Xenopus as a model to<br />
characterise the role <strong>of</strong> IGFBP5 in cardiac development. Overexpression <strong>of</strong> IGFBP5 mRNA on the left side <strong>of</strong><br />
the two-cell stage embryo resulted in 22% abnormal A or L looping (p
increase <strong>of</strong> hoxb5b at least in part causes the increase in the CM number. In contrast to inhibiti ng RA signaling<br />
components using other methods, examination <strong>of</strong> additional RA responsive genes and RARs indicates that their<br />
expression is also increased in RARαb1 deficient embryos, suggesting that the loss <strong>of</strong> RARαb1 results in a<br />
general increase <strong>of</strong> RA signaling. Although effects were not observed on the development <strong>of</strong> the heart size or<br />
CM number, similar effects on RA signaling components were found in RARαb2 deficient embryos. Altogether,<br />
our results suggest an intriguing model where depletion <strong>of</strong> RARαb1 results in a feedback loop that induces<br />
modest increases in RA signaling, which ultimately promote increased CM specification.<br />
6.17 Coordinated myocardial movements during progressive stages <strong>of</strong> heart tube assembly and early<br />
looping.<br />
1 Aleksandrova, Anastasiia; 2 Lansford, Rusty; 1 Little, Charles D.; 1 Czirok, Andras; 1 Rongish, Brenda J.<br />
1 Department <strong>of</strong> Anatomy and Cell Biology, <strong>University</strong> <strong>of</strong> Kansas Medical Center, 3901 Rainbow Blvd. Kansas<br />
City, KS 66160 and 2 Biological Imaging Center, California Institute <strong>of</strong> Technology, 1200 E California, Pasadena,<br />
CA 91125<br />
Transformation <strong>of</strong> planar cardiac progenitor fields into a progressively more complex structure, the heart,<br />
involves a series <strong>of</strong> cellular and tissue movements in three dimensions. However, the driving forces behind<br />
these movements are not fully understood. We investigated the motion <strong>of</strong> fluorescently tagged myocardial<br />
progenitors in live quail embryos using time-lapse imaging. The fibronectin ECM environment for myocardial<br />
movement was visualized in vivo with microinjected fluorescent antibodies, while endocardial progenitors were<br />
labeled with transgenic expression <strong>of</strong> Tie1::H2B-YFP. The imaging period encompassed the motion <strong>of</strong><br />
myocardial progenitors from primary heart field(s) to the midline and continued through early heart looping<br />
stages. We determined the relative importance <strong>of</strong> directed cell autonomous (relative to the ECM) motility versus<br />
convective tissue movements in the assembly <strong>of</strong> a myocardial layer <strong>of</strong> the cardiac tube using object tracing and<br />
particle image velocimetry. These quantitative data indicate the contribution <strong>of</strong> cell autonomous motility<br />
displayed by myocardial progenitors is limited, with convective tissue movements playing a major role in their<br />
arrival at the midline. Further, concomitantly with the onset <strong>of</strong> cardiac tube bending, caudal myocardial primordia<br />
underwent a coordinated displacement in the posterior-ventral direction (“rotation”), suggesting a possible<br />
connection between these processes. Interestingly, this rotation was abrogated following the introduction <strong>of</strong><br />
exogenous VEGF165, but proceeded in separated primordia following incision-induced cardia bifida. Ongoing<br />
studies are underway to assess the effects <strong>of</strong> myocardial-specific expression <strong>of</strong> dominant negative and<br />
constitutively active is<strong>of</strong>orms <strong>of</strong> RhoA on rotation and to further elucidate the cellular and tissue mechanisms<br />
driving this bulk movement.<br />
6.18 Primary cilia control proliferation <strong>of</strong> myocardium during early cardiogenesis<br />
Christopher Lin, Sherry Zhou, Martina Brueckner<br />
Yale <strong>University</strong> School <strong>of</strong> Medicine, Dept <strong>of</strong> Pediatrics and Genetics<br />
Cardiac contraction influences morphogenesis, however, the sensing mechanism remains unclear. Cardiac cilia<br />
are found in the embryonic mouse heart, and embryos lacking cilia have thinned compact myocardium. We<br />
evaluated myocardial proliferation in mouse embryos with mutations affecting ciliogenesis: Kif3A-/- and IFT20-/-.<br />
Hearts were isolated from 11-20somite stage embryos, mitotic cells labeled with anti-Phospho histone, imaged<br />
using optical sectioning and total and PH3+ cells were counted. At 11 somites, cell counts in WT, Kif3A-/- and<br />
IFT20-/- are the same. By 20 somite stage Kif3A-/- and IFT20-/- hearts have 30% fewer cells than WT. PH3<br />
staining shows that the percent PH3 positive myocardial cells is unchanged between WT, Kif3A-/- and IFT20-/-.<br />
However, the ratio <strong>of</strong> myocardial cells in prophase, metaphase and anaphase differed significantly: Kif3A-/- and<br />
IFT20-/- embryo hearts had significantly fewer myocardial cells that had progressed from prophase to<br />
metaphase and anaphase than WT. These data indicate that failure <strong>of</strong> myocardial cells to progress through the<br />
cell cycle contributes to myocardial thinning in embryos with defective cilia. The proliferative defects observed in<br />
Kif3A-/- and IFT20-/- hearts coincided with onset <strong>of</strong> contraction. To evaluate the effect <strong>of</strong> contraction on<br />
myocardial proliferation, we evaluated hearts <strong>of</strong> Ncx-/- embryos, which do not contract. Cell counts and PH3<br />
staining in the Ncx-/- hearts were indistinguishable from cilia-defective hearts. These data suggest that<br />
contraction provides a signal for myocardial proliferation, and support the hypothesis that cilia in the<br />
myocardium may function as sensors for contraction, and that they have a role in control <strong>of</strong> myocardial<br />
proliferation.<br />
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6.19 Functional significance <strong>of</strong> SRJ domain mutations in CITED2<br />
Chiann-mun Chen 1,2 , Jamie Bentham 1,2 , Catherine Cosgrove 1,2 , Jose Braganca 1,2,3 , Simon D. Bamforth 1,2,4 ,<br />
Jürgen E. Schneider 1,2 , Hugh Watkins 1,2 , Bernard Keavney 4 , Benjamin Davies 2 and Shoumo Bhattacharya 1,2<br />
1 Department <strong>of</strong> Cardiovascular Medicine, 2 Wellcome Trust Centre for Human Genetics, <strong>University</strong> <strong>of</strong> Oxford,<br />
Roosevelt Drive, Oxford, OX3 7BN, UK, 3 Institute for Biotechnology and Bioengineering, Centre for Molecular<br />
and Structural Biomedicine (CBME), <strong>University</strong> <strong>of</strong> Algarve, Faro, Portugal, 4 Institute <strong>of</strong> Genetic Medicine,<br />
Newcastle <strong>University</strong>, Newcastle, UK.<br />
CITED2 is a transcriptional co-activator with 3 conserved domains shared with other CITED family members<br />
and a unique Serine-Glycine Rich Junction (SRJ) that is highly conserved in placental mammals. Loss <strong>of</strong> Cited2<br />
in mice results in cardiac and aortic arch malformations, adrenal agenesis, neural tube and placental defects,<br />
and partially penetrant defects in left-right patterning. Non-synonymous mutations in CITED2 have previously<br />
been detected in small studies <strong>of</strong> human congenital heart disease (CHD). By screening 1126 sporadic CHD<br />
cases and 1227 controls, we identified 19 variants, including 5 unique non-synonymous sequence variations<br />
(T166N, R92G, N62S and A187T, G180-A187del) in patients. Many <strong>of</strong> the CHD-specific variants identified in<br />
this and previous studies cluster in the SRJ domain. Transient transfection experiments indicated that T166N<br />
mutation impaired TFAP2 co-activation function. In order to investigate the functional significance in vivo, we<br />
generated a T166N mu tation <strong>of</strong> mouse Cited2. We also used PhiC31 integrase-mediated cassette exchange to<br />
generate a Cited2 knock-in allele replacing the mouse Cited2 coding sequence with human CITED2 and with a<br />
mutant form deleting the entire SRJ domain. Mouse embryos expressing only CITED2-T166N or CITED2-SRJdeleted<br />
alleles surprisingly show no morphological abnormalities, and mice are viable and fertile. These results<br />
indicate that the SRJ domain is dispensable for these functions <strong>of</strong> CITED2 in mice and that point mutations and<br />
deletions clustering in the SRJ region are unlikely to be the sole cause <strong>of</strong> the malformations observed in patients<br />
with sporadic CHD. We suggest that additional factors are required to cause CHD in such patients.<br />
6.20 Distinct phases <strong>of</strong> Wnt/ß-catenin signaling direct cardiomyocyte formation in zebrafish.<br />
Dohn, TE 1, 2 , Waxman, JS 2 .<br />
1 Molecular and Developmental Biology Graduate Program, <strong>University</strong> <strong>of</strong> Cincinnati and Cincinnati Children’s<br />
Hospital. 2 The Heart Institute and Molecular Cardiovascular Biology Division, Cincinnati Children’s Medical<br />
Center, Cincinnati, OH, USA<br />
Normal heart formation requires distinct phases <strong>of</strong> canonical Wnt/β-catenin (Wnt) signaling. Understanding the<br />
mechanisms by which Wnt signaling drives correct cardiac formation in vivo is critical for understanding the<br />
formation <strong>of</strong> the heart as well as essential to studies developing stem cells into cardiomyocytes (CM) in vitro.<br />
Here, we investigated the roles <strong>of</strong> Wnt signaling using heat-shock inducible transgenes which allow us to<br />
increase or decrease Wnt signaling in the embryo. During the first 24 hours <strong>of</strong> development, we find three<br />
distinct phases during which Wnt signaling modulates CM formation. Wnt signaling has previously been<br />
implicated in mesoderm specification as well as regulating the pre-cardiac mesoderm. Here, we have also<br />
identified a later role after CM differentiation has initiated, in which Wnt signaling is necessary and sufficient to<br />
promote the differentiation <strong>of</strong> atrial cells. This study also extends the previous studies on Wnt signaling during<br />
mesoderm specification and in the pre-cardiac mesoderm in zebrafish. Interestingly, we define a new role for<br />
Wnt signaling in the pre-cardiac mesoderm where Wnt is sufficient to prevent cardiac cell differentiation, which<br />
leads to Caspase-3 independent cell death. Together with a previously described later role <strong>of</strong> Wnt signaling in<br />
restricting ventricular CM proliferation, our results indicate that there are four distinct phases <strong>of</strong> Wnt signaling<br />
during the first 3 days <strong>of</strong> zebrafish development that allow for the proper formation <strong>of</strong> the heart.<br />
6.21 Defining Paraxial mesoderm contribution to the heart in chick embryo<br />
Radwan Abu-Issa, Ph.D.<br />
<strong>University</strong> <strong>of</strong> Michigan-Dearborn, MI. USA.<br />
The myocardium is derived from the splanchnic mesoderm sheet (SMS) directly where the endocardium is<br />
derived from cells that delaminate first from the SMS towered the endoderm sheet and then fuse medially<br />
forming a tube. The SMS moves as a sheet and fuse medially around stage HH9-10, it continues till the entire<br />
myocardium is formed around stage HH18-19, around stage HH12 the middle <strong>of</strong> the sheet <strong>of</strong> the right and left<br />
side fuse together dorsally giving rise for the first time to the formation <strong>of</strong> the heart tube, from now on the<br />
contribution to the two heart poles (outflow and inflow) becomes independent. The SMS continues laterally with<br />
paraxial mesoderm and so the question is how much if any <strong>of</strong> the paraxial mesoderm does contribute to the<br />
myocardium. Using crystal dye that allow lebeling for few cells diameter at one time preventing unwanted<br />
143
diffusion but can label hundreds <strong>of</strong> cells that pass by over longer time, cells <strong>of</strong> the paraxial mesoderm at<br />
different distances from SMS a nd at different stages (HH8-HH13) where labeled and followed till later stages<br />
(HH18-19) and their contribution where visualized either by cryo-sectioning or special clearing <strong>of</strong> the embryo.<br />
The results unequivocally demonstrate that in addition <strong>of</strong> the splanchnic mesoderm, a small adjacent group <strong>of</strong><br />
cells (5-6 cells diameter) behave like a source and contribute quite consistently to the forming myocardium with<br />
the complete embryonic ventral closure.<br />
6.22 Mouse-chick chimeric transplantation reveals the regional contribution <strong>of</strong> the cell population<br />
arising from the inflow tract to cardiac chamber formation<br />
Rieko Asai 1,2 , Yukiko Kurihara 1 , Yuichiro Arima 1 , Ki-Sung Kim 1 , Sachiko Miyagawa-Tomita 2 , Hiroki Kurihara 1<br />
1<br />
Dept. <strong>of</strong> Phys. Chem. and Metabol., Grad. Sch. <strong>of</strong> Med., Univ. <strong>of</strong> Tokyo<br />
2<br />
Medical Research Institute,Pediatric Cardiology, TWMU<br />
Previously, we identified a distinct cell population defined by the expression <strong>of</strong> Endothelin type-A receptor<br />
(Ednra) in the mouse early inflow region. This population mainly contributes to the left ventricle and atria during<br />
E8.25 to E9.5. To facilitate lineage analysis <strong>of</strong> this cell population during cardiac development, we established a<br />
mouse-chick chimera model. Cardiac inflow tissues corresponding to Ednra-expressing region were excised<br />
from E8.25 (5- to 7-somite stage) mouse embryos carrying the Mesp1-Cre;R26R reporter gene and transplanted<br />
into the same regions <strong>of</strong> 2-day-old chick embryos without removing their own tissues. After 3 days, bgalactosidase-positive<br />
cells derived from the mouse graft were detected in the chick heart, especially in the left<br />
ventricle, atria, venous valves and ventricular septum. In the left ventricle, b-galactosidase /myosin heavy chain<br />
(MHC) double-positive cells were frequently observed. The distribution patterns <strong>of</strong> b-galactosidase-positive cells<br />
appeared to partially reflect similar to those <strong>of</strong> Ednra-expressing cells. In addition, in vitro culture showed Ednra-<br />
EGFP-labeled colonies expressing MHC arising from the inflow region. These results indicate the regional<br />
contribution <strong>of</strong> the inflow cell population characterized by Ednra expression to chamber formation. This mousechick<br />
chimera system is useful for lineage tracing and fate mapping <strong>of</strong> genetically-labeled mouse cardiogenic<br />
tissues.<br />
6.23 Identification <strong>of</strong> a Novel Developmental Mechanism in the Generation <strong>of</strong> Mesothelia.<br />
Nichelle Winters 1 , Rebecca Thomason 1 , David Bader 1,2<br />
1<br />
Department <strong>of</strong> Cell and Developmental Biology, Vanderbilt <strong>University</strong><br />
2<br />
Department <strong>of</strong> Medicine, Vanderbilt <strong>University</strong><br />
Mesothelial cells form the surface layer <strong>of</strong> all coelomic structures and are essential to organ function. During<br />
development, these cells undergo an epithelial to mesenchymal transition (EMT) to provide the precursors for<br />
the vasculature and stromal cells to all coelomic organs investigated to date. Furthermore, in the adult,<br />
mesothelial cells stimulated by disease or injury retain the ability to undergo EMT to generate fibroblasts and<br />
vascular smooth muscle cells mimicking their developmental behavior. Despite the broad contribution <strong>of</strong> this cell<br />
type to developing organs and adult disease, our current understanding <strong>of</strong> the genesis <strong>of</strong> this cell type is<br />
confined to a single organ, the heart, in which an exogenous population <strong>of</strong> cells, the proepicardium, migrates to<br />
and over the myocardium to give rise to the cardiac mesothelium and coronary vasculature. It is unknown<br />
whether this pattern <strong>of</strong> development is specific to the heart or applies broadly to other coelomic organs. Using<br />
two independent long term lineage tracing studies, we demonstrate that mesothelial progenitors <strong>of</strong> the intestine<br />
are intrinsic to the gut tube anlage. Furthermore, a novel chick-quail chimera model <strong>of</strong> gut morphogenesis<br />
reveals these mesothelial progenitors are broadly distributed throughout the gut primordium and are not derived<br />
from a localized and exogenous proepicardium-like source <strong>of</strong> cells. These data demonstrate an intrinsic origin <strong>of</strong><br />
mesothelial cells to a coelomic organ and provide a novel mechanism for the generation <strong>of</strong> mesothelial cells.<br />
6.24 Characterization <strong>of</strong> the pro-cardiac activity conferred by Gata5 and Smarcd3b in the zebrafish<br />
embryo<br />
Ashish R. Deshwar and Ian C. Scott<br />
Program in Developmental and Stem Cell Biology, The Hospital for Sick Children and Department <strong>of</strong> Molecular<br />
Genetics, <strong>University</strong> <strong>of</strong> Toronto, Toronto, ON, Canada<br />
In the zebrafish cardiac progenitors are first found in the blastula in several rows <strong>of</strong> cells at the embryonic<br />
margin. During gastrulation these cells ingress and move to the anterior lateral plate mesoderm (ALPM) where<br />
they will then migrate towards the midline and fuse to form a heart tube. How cardiac progenitors specifically<br />
migrate to the ALPM, how they become specified to the cardiac lineage and how they are maintained are all<br />
144
questions that remain unanswered. The transcription factor Gata5 and chromatin re-modeling complex sub-unit<br />
Smarcd3b are able to direct non-cardiac cells to migrate to the ALPM and form part <strong>of</strong> the heart in the<br />
developing zebrafish embryo. Studying how this pro-cardiac activity is accomplished may help address some <strong>of</strong><br />
these outstanding questions. A conditional version <strong>of</strong> Gata5 been utilised to determine the temporal requirement<br />
<strong>of</strong> it in this phenomena. Gata5 activity is found to be necessary early in gastrulation for pro-cardiac activity which<br />
may suggest a novel role for it in heart development. In order to fully characterize the molecular mechanisms<br />
which underlie this activity transcriptional pr<strong>of</strong>iling has been performed on gata5/smarcd3b over-expressing cells<br />
during gastrulation. This has revealed a unique set <strong>of</strong> transcripts in these cells <strong>of</strong> which further analysis may<br />
reveal novel regulators <strong>of</strong> cardiac progenitor development.<br />
6.25 Interrogating Cardiovascular Morphogenesis in Zebrafish Through Small Molecule Perturbation<br />
Grant I. Miura, Richard Shehane, David L. Stachura, David Traver, and Deborah Yelon<br />
Division <strong>of</strong> Biological Sciences, <strong>University</strong> <strong>of</strong> California, San Diego, La Jolla, CA, 92093, USA<br />
Cardiogenesis involves the coordination <strong>of</strong> complex cell behaviors necessary to generate the three-dimensional<br />
structure <strong>of</strong> the heart. For example, heart tube assembly requires the migration <strong>of</strong> bilateral populations <strong>of</strong><br />
cardiomyocytes to the midline where they meet and merge to form a cardiac ring through a process called<br />
cardiac fusion. Several genes are known to influence cardiac fusion, yet our understanding <strong>of</strong> the regulation <strong>of</strong><br />
this aspect <strong>of</strong> cardiac morphogenesis remains incomplete. We aim to identify additional mediators <strong>of</strong> cardiac<br />
fusion by screening for small molecules that can alter the dimensions <strong>of</strong> the cardiac ring. So far, our pilot screen<br />
has identified four compounds <strong>of</strong> interest, and we have focused on two, ketanserin and scopoletin, that expand<br />
the shape <strong>of</strong> the cardiac ring. Treatment with ketanserin, a serotonin receptor antagonist, and treatment with<br />
scopoletin, a coumarin derivative, both seem to disrupt cell movements during cardiac fusion, resulting in<br />
defects in myocardial and endocardial tube assembly. In addition, ketanserin treatment disrupts atrioventricular<br />
canal differentiation, whereas scopoletin treatment leads to an increased number <strong>of</strong> erythrocytes. Therefore, we<br />
propose that ketanserin and scopoletin disrupt two distinct pathways with similar influences on cardiac fusion.<br />
We are currently investigating the molecular mechanisms by which ketanserin and scopoletin affect<br />
cardiovascular development.<br />
6.26 Snx30: a new negative regulator <strong>of</strong> Wnt/β-catenin signaling identified in the frog.<br />
Cameron M 1, 2 , Leclerc S 1 , Chemtob S 1, 2 1, 2<br />
, Andelfinger A<br />
1<br />
CHU Sainte-Justine, Montreal, Quebec, Canada<br />
2<br />
Pharmacology department, <strong>University</strong> <strong>of</strong> Montreal, Montreal, Quebec, Canada<br />
Wnt/β-catenin signaling is a key regulator <strong>of</strong> cardiogenesis that occurs in distinct phases <strong>of</strong> activation and<br />
repression. Although secreted antagonists <strong>of</strong> Wnt/β-catenin signaling have been identified, intracellular<br />
regulation <strong>of</strong> this pathway is poorly understood. In this study, we identified Snx30, a new repressor <strong>of</strong> Wnt/βcatenin<br />
signaling required during early cardiac specification in Xenopus laevis. Snx30 is a member <strong>of</strong> the<br />
sorting nexin family that regulate intracellular trafficking <strong>of</strong> membrane-bound proteins. In Xenopus embryos,<br />
Snx30 was expressed in three phases, the strongest peak occuring before gastrulation and limited to the animal<br />
pole <strong>of</strong> the blastula. This triphasic pattern <strong>of</strong> expression was similar to that observed in differentiating human<br />
cardiomyocytes. Also, expression in human fetal heart was at least two-fold stronger than in adult heart.<br />
Knockdown <strong>of</strong> Snx30 in Xenopus embryos caused a broad phenotype, with 60% mortality, developmental delay<br />
appearing at the onset <strong>of</strong> gastrulation and generalized anterior defects. A closer look revealed upregulation <strong>of</strong><br />
classical Wnt/β-catenin target genes, Xnr3 and Siamois, as well as cardiac defects. Data from<br />
immunohistochemistry and subcellular fractionation in HEK293 cells suggest that SNX30 localizes to early<br />
endosomes. In conclusion, we propose that Snx30 functions as a repressor <strong>of</strong> Wnt/β-catenin signaling during<br />
the inhibitory phases required for early cardiac specification. This role may be accomplished by trafficking <strong>of</strong><br />
Wnt receptors towards an intracellular degradation route, as seen in other sorting nexins, but this requires<br />
further study. Understanding the early steps <strong>of</strong> cardiogenesis could help develop better techniques to produce<br />
functional cardiomyocytes used in stem cell therapy.<br />
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6.27 Vascular Endothelial And Endocardial Progenitors Differentiate As Cardiomyocytes In The<br />
Absence Of Etsrp/Etv2 Function<br />
Sharina Palencia-Desai 1,2 , Vikram Kohli 1 , Jione Kang 3 , Neil C. Chi 4 , Brian L. Black 3 and Saulius Sumanas 1,2 ,*<br />
1 Division <strong>of</strong> Developmental Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229, USA.<br />
2 <strong>University</strong> <strong>of</strong> Cincinnati College <strong>of</strong> Medicine, Cincinnati, OH 45229, USA. 3 Department <strong>of</strong> Biochemistry and<br />
Biophysics, <strong>University</strong> <strong>of</strong> California, San Francisco, CA 94158 USA. 4 Department <strong>of</strong> Medicine, Division <strong>of</strong><br />
Cardiology, <strong>University</strong> <strong>of</strong> California, San Diego School <strong>of</strong> Medicine, San Diego, CA 92093, USA.<br />
Previous studies have suggested that embryonic vascular endothelial, endocardial and myocardial lineages<br />
originate from multipotential cardiovascular progenitors. However, their existence in vivo has been debated and<br />
molecular mechanisms that regulate specification <strong>of</strong> different cardiovascular lineages are poorly understood. An<br />
ETS domain transcription factor Etv2/Etsrp/ER71 has been recently established as a crucial regulator <strong>of</strong><br />
vascular endothelial differentiation in zebrafish and mouse embryos. In this study, we show that etsrpexpressing<br />
vascular endothelial/endocardial progenitors differentiate as cardiomyocytes in the absence <strong>of</strong> Etsrp<br />
function during zebrafish embryonic development. Expression <strong>of</strong> multiple endocardial specific markers is absent<br />
or greatly reduced in Etsrp knockdown or mutant embryos. We show that Etsrp regulates endocardial<br />
differentiation by directly inducing endocardial nfatc1 expression. In addition, Etsrp function is required to inhibit<br />
myocardial differentiation. In the absence <strong>of</strong> Etsrp function, etsrp-expressing endothelial and endocardial<br />
progenitors initiate myocardial marker hand2 and cmlc2 expression. Furthermore, Foxc1a function and<br />
interaction between Foxc1a and Etsrp is required to initiate endocardial development, but is dispensable for the<br />
inhibition <strong>of</strong> myocardial differentiation. These results argue that Etsrp initiates endothelial and endocardial, and<br />
inhibits myocardial, differentiation by two distinct mechanisms. Our findings are important for the understanding<br />
<strong>of</strong> genetic pathways that control cardiovascular differentiation during normal vertebrate development and will<br />
also greatly contribute to the stem cell research aimed at regenerating heart tissues.<br />
6.28 Nkx2-5 mediates differential cardiac differentiation through interaction with Hoxa10<br />
Ann N. Behrens 1 , Michelina Iacovino 1,3 , Jamie L. Lohr 1 , Yi Ren 1 , Claudia Zierold 1 , Richard P. Harvey 4 , Michael<br />
Kyba 1,3 , Daniel J. Garry 1,2 and Cindy M. Martin 1,2<br />
1 Lillehei Heart Institute, <strong>University</strong> <strong>of</strong> Minnesota, Minneapolis, Minnesota 2 Department <strong>of</strong> Internal Medicine,<br />
<strong>University</strong> <strong>of</strong> Texas Southwestern Medical Center, Dallas, Texas. 3 Department <strong>of</strong> Developmental Biology,<br />
<strong>University</strong> <strong>of</strong> Texas Southwestern Medical Center, Dallas, Texas. 4 Victor Chang Cardiac Research Institute,<br />
Lowy Packer Building, 405 Liverpool St., Darlinghurst 2010, Australia; and St. Vincent’s Clinical School,<br />
<strong>University</strong> <strong>of</strong> New South Wales, Kensington 2052, Australia.<br />
Nkx2-5 has long been utilized as a marker <strong>of</strong> early cardiac progenitor cells. Recent studies have documented<br />
that Nkx2-5 positive cells are not limited to the cardiac lineage but can give rise to endothelial and smooth<br />
muscle lineages. Other work has elucidated that, in addition to promoting cardiac development, Nkx2-5 plays a<br />
larger role in mesodermal patterning although the transcriptional networks that govern this developmental<br />
patterning are unclear. By pr<strong>of</strong>iling early Nkx2-5 positive progenitor cells, we discovered the progenitor pools <strong>of</strong><br />
the bisected cardiac crescent are differentiating asynchronously and the relative acceleration <strong>of</strong> the left side is<br />
Nkx2-5 dependent. Surprisingly, posterior Hox genes, Hoxa9 and Hoxa10, were expressed in the right side,<br />
independently <strong>of</strong> Nkx2-5. Hoxa10 has been shown to play a role in regulating hematopoietic proliferation and<br />
differentiation but posterior Hox genes have never been implicated in early mammalian cardiac development. In<br />
differentiating ES cells, Hoxa10 is expressed in the earliest Nkx2-5 positive progenitors and ectopic Hoxa10<br />
expression reduces structural cardiac gene expression. This data suggests a model in which Nkx2-5 mediates<br />
differential timing <strong>of</strong> cardiac differentiation through an unprecedented interaction with a posterior Hox gene.<br />
6.29 Selective loss <strong>of</strong> Vcan Proteins Causes Heart Defects and Alters Collagen Deposition.<br />
Mjaatvedt, Corey H 2 , Le Saux, Claude Jourdan 1 , Schwacke, John H 2 , Davis, Monica A 2 and Krug, Edward L 2<br />
1 Dept. <strong>of</strong> Medicine/Division <strong>of</strong> Cardiology, UTHSCSA, TX, USA 78229. 2 Dept. <strong>of</strong> Regenerative Medicine,<br />
Medical <strong>University</strong> <strong>of</strong> S. Carolina, Charleston, SC, USA 29464-8150<br />
The requirement for the proteoglycan versican (Vcan) to early heart formation was clearly demonstrated by the<br />
Vcan null mouse called heart defect (hdf). Total absence <strong>of</strong> the Vcan gene is lethal at a stage prior to the<br />
heart’s pulmonary/aortic outlet segment growth. This creates a challenge for determining Vcan’s significance in<br />
the forming valve precursors and vascular wall <strong>of</strong> the pulmonary and aortic trunks. Fortunately, 3 mouse models<br />
<strong>of</strong> partial Vcan depletion do survive to birth. Both the hdf hemizygous and halpn1 null mice have reduced (50%)<br />
levels <strong>of</strong> Vcan expression and similar heart defects. The third model, a deletion <strong>of</strong> exon 7 in the Vcan gene also<br />
146
has reduced levels (50%) <strong>of</strong> Vcan expression due to the complete lack <strong>of</strong> two <strong>of</strong> the 4 alternative Vcan forms.<br />
Comparison <strong>of</strong> the 3 models showed similarities in heart defects such as, ventricular septal defects and a thin<br />
myocardium. However, the exon 7 null also has an additional spectrum <strong>of</strong> cardiac outlet defects that include<br />
changes in valve precursors and walls <strong>of</strong> the pulmonary and aortic trunks. We compare the changes in E13.5<br />
and neonate hearts by biochemical and histological analysis including Hsp47 western blot, sircol quantification,<br />
and pircrosirius red staining patterns. A change in abundance and banding pattern <strong>of</strong> the collagen fibril<br />
chaperone protein, Hsp47 was measured. Hearts lacking Vcan exon 7 have a 2.5 fold reduction <strong>of</strong> insoluble<br />
compared to soluble collagen (p
S7.2 Zebrafish second heart field development relies on early specification <strong>of</strong> progenitors and nkx2.5<br />
function.<br />
Burcu Guner-Ataman 1 , Noelle Paffett-Lugassy 1,2 , Meghan S. Adams 1 , Kathleen R. Nevis 1 , Caroline E. Burns 1,2 ,<br />
C. Ge<strong>of</strong>frey Burns 1<br />
1 2<br />
Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, MA 02129; Harvard Stem<br />
Cell Institute, Cambridge, MA, 02138<br />
We recently reported that latent TGFβ binding protein 3 (ltbp3) marks the zebrafish anterior second heart field<br />
(SHF) that contributes myocardium to the distal ventricle and three cardiovascular lineages to the outflow tract<br />
(OFT). However, SHF expression <strong>of</strong> ltbp3 initiates at the arterial pole <strong>of</strong> the linear heart tube, well after higher<br />
vertebrates specify SHF progenitors in the lateral plate mesoderm (LPM). Building on a recent dye tracing study<br />
from the Kirby laboratory, we identified putative nkx2.5+ SHF progenitors in the zebrafish anterior LPM (ALPM),<br />
cranial and medial to adjacent early-differentiating cardiomyocytes (nkx2.5+, cmlc2+). Using inducible Cre/loxPmediated<br />
lineage tracing, we pulse-labeled nkx2.5+, GATA4+, or cmlc2+ cells in the ALPM and characterized<br />
their descendents using lineage specific reporters. From these analyses, we learned that pulse-labeled nkx2.5+<br />
and GATA4+ cells give rise to myocardium in the entire ventricle and three cardiovascular li neages in the OFT.<br />
Pulse-labeling <strong>of</strong> cmlc2+ cells revealed that early differentiating cardiomyocytes reside predominantly in the<br />
proximal ventricle. Taken together, these data suggest strongly that nkx2.5+, GATA4+, cmlc2- SHF progenitors<br />
are specified in the zebrafish ALPM prior to initiating expression <strong>of</strong> ltbp3 at the arterial pole. Furthermore, we<br />
tested whether nkx2.5 plays a conserved and essential role during zebrafish SHF development. Nkx2.5<br />
morphant embryos exhibited several SHF-related phenotypes including significant reductions in distal ventricular<br />
myocardium, outflow tract smooth muscle, and expression <strong>of</strong> ltbp3. Taken together, our data reveal two<br />
conserved features <strong>of</strong> zebrafish SHF development underscoring the utility <strong>of</strong> this model organism for<br />
deciphering SHF biology.<br />
S7.3 Notch1 mediated signaling cascades regulate cardiomyocyte polarity and ventricular wall<br />
formation.<br />
1 1 2 1 2 2 1<br />
Wenjun Zhang, Hanying Chen, Momoko Yoshimoto, Jin Zhang, Mervin C Yoder, Nadia Carlesso, Weinian<br />
Shou<br />
1<br />
Riley Heart Research Center, Herman B Wells Center for Pediatric Research, Department <strong>of</strong> Pediatrics,<br />
Indiana <strong>University</strong> School <strong>of</strong> Medicine. 2 Division <strong>of</strong> Neonatology, Herman B. Wells Center for Pediatric<br />
Research, Department <strong>of</strong> Pediatrics, Indiana <strong>University</strong> School <strong>of</strong> Medicine, Indianapolis<br />
Ventricular trabeculation and compaction are two anatomically overlapping morphogenetic events essential for<br />
normal myocardial development. Disregulation <strong>of</strong> these events can lead to Left Ventricular Noncompaction<br />
(LVNC, MIM300183). However, the underlying mechanisms and pathogenetic pathways remain elusive, largely<br />
due to the genetic heterogeneity <strong>of</strong> the patients and the lack <strong>of</strong> suitable animal models. Recently, through the<br />
use <strong>of</strong> gene pr<strong>of</strong>iling and cardiac cell-lineage restricted genetic manipulation, we analyzed a unique mouse<br />
genetic model for LVNC, Fkbp1a knockout mice. Our data demonstrated a critical contribution <strong>of</strong> over-activated<br />
Notch1 and subsequent neuregulin1-ErbB2/4 signaling in developing LVNC in mouse, and confirmed previous<br />
finding that Notch1-neuregulin1/Bmp10 signaling pathways in regulating ventricular cardiomyocyte proliferation.<br />
Remarkably, our new emerging data also suggested that the excessively activated Notch1-neuregulin1/ErbB2<br />
signaling cascade dramatically down-regulated dishevelled-associated activator <strong>of</strong> morphogenesis 1 (Daam1), a<br />
member <strong>of</strong> the Formin family and a potential effector <strong>of</strong> non-canonical Wnt planer cell polarity (PCP) signaling.<br />
Genetic ablation <strong>of</strong> Daam1 resulted in disoriented cardiomyocyte polarity with altered actin cytoskeleton and<br />
my<strong>of</strong>iber organization, and concomitantly ventricular noncompaction, which confirmed the role <strong>of</strong> Daam1 in the<br />
development <strong>of</strong> LVNC. More interestingly, we have also identified that p21-activated kinase 1 (Pak1), a Ser/Thr<br />
protein kinase known to control tyrosine kinase receptor (i.e., ErbB2/4) phosphorylation and small GTPases<br />
activities in regulating cell proliferation, cell polarity, and actin cytoskeleton organization, is a potential up-stream<br />
regulator <strong>of</strong> Daam1. Activated Pak1 (phospho-T423) was dramatically up-regulated in Fkbp1a mutant hearts<br />
with excessive activation <strong>of</strong> ErbB2/4. In addition, over-expression <strong>of</strong> constitutively active Pak1 (Pak1ca) in<br />
primary cultured neonatal cardiomyocytes down-regulated the level <strong>of</strong> Daam1. Taken together, our in vivo and<br />
in vitro data demonstrated that Notch-neuregulin-Pak1-Daam1 is an essential signaling cascade in regulating<br />
cardiomyocyte polarity and ventricular wall formation.<br />
148
S7.4 Mutations in the NOTCH pathway regulator MIB1 cause ventricular non-compaction<br />
cardiomyopathy<br />
Guillermo Luxán 1 , Jesús C. Casanova 1 , Beatriz Martínez-Poveda 1 , Donal MacGrogan 1 , Álvaro Gonzalez-Rajal 1 ,<br />
Belén Prados 1 , Gaetano D'Amato 1 , David Dobarro 1 , Carlos Torroja 2 , Fernando Martinez 2 , José Luis Izquierdo-<br />
García 3 , Leticia Fernández-Friera 3 , María Sabater-Molina 4 , Young-Y. Kong 5 , Gonzalo Pizarro 3 , Borja Ibañez 3 ,<br />
Constancio Medrano 6 , Pablo García-Pavía 7 , Juan R. Gimeno 4 , Lorenzo Montserrat 8 , Luis J. Jiménez-<br />
Borreguero 3 & José Luis de la Pompa 1<br />
1 Program <strong>of</strong> Cardiovascular Developmental Biology, Department <strong>of</strong> Cardiovascular Development and Repair, Centro<br />
Nacional de Investigaciones Cardiovasculares (CNIC), Melchor Fernández Almagro 3, 28029 Madrid, SPAIN.<br />
2 Bioinformatics Unit, CNIC, Melchor Fernández Almagro 3, 28029 Madrid SPAIN<br />
3 Department <strong>of</strong> Epidemiology, Atherothrombosis and Image, CNIC, Melchor Fernández Almagro 3, 28029 Madrid SPAIN.<br />
4 Hospital Universitario Virgen de la Arrixaca, Ctra. Murcia-Cartagena s/n 30120 El Palmar, Murcia, SPAIN. 5 Department <strong>of</strong><br />
Biological Sciences, Seoul National <strong>University</strong>, Seoul, Republic <strong>of</strong> Korea. 6 Department <strong>of</strong> Cardiology, Hospital General<br />
Universitario Gregorio Marañón, Madrid, SPAIN. 7 Cardiomyopathy Unit. Department <strong>of</strong> Cardiology. Hospital Universitario<br />
Puerta de Hierro, Manuel de Falla 1, 28222 Majadahonda SPAIN. 8 Instituto de Investigación Biomédica de A Coruña,<br />
Complejo Hospitalario Universitario A Coruña, As Xubias 84, 15006 A Coruña, SPAIN<br />
Ventricular non-compaction (VNC) cardiomyopathy patients show prominent trabeculations mainly in the left<br />
ventricle and reduced systolic function. Clinical presentation varies from asymptomatic to heart failure. We show<br />
that germline mutations in human MIND BOMB-1 (MIB1), encoding an E3 ubiquitin ligase that promotes<br />
endocytosis <strong>of</strong> the NOTCH ligands DELTA and JAGGED, cause VNC in autosomal-dominant pedigrees, and<br />
affected individuals show reduced NOTCH1 activity. Functional studies in cells and zebrafish embryos and in<br />
silico modeling indicate that these are loss-<strong>of</strong>-function phenotypes. VNC is triggered by targeted inactivation <strong>of</strong><br />
Mib1 in mouse myocardium, and mimicked by inactivation <strong>of</strong> myocardial Jagged1 or endocardial Notch1.<br />
Myocardium-specific Mib1 mutants show reduced ventricular Notch1 activity, and an expansion <strong>of</strong> compact<br />
myocardium markers to the abnormally proliferative, immature trabeculae. These results identify NOTCH as a<br />
primary signaling pathway involved in VNC, and suggest that MIB1 mutations cause a developmental arrest in<br />
chamber myocardium, preventing trabecular maturation and compaction.<br />
S7.5 The early role <strong>of</strong> Tbx1 in anterior and posterior second heart field cells<br />
M. Sameer Rana 1 , Karim Mesbah 2 , Jorge N. Dominguez 3 , Niels H<strong>of</strong>schreuder 1 , Virginia E. Papaioannou 4 ,<br />
Antoon F. Moorman 1 , Vincent M. Christ<strong>of</strong>fels 1 , Robert G. Kelly 2<br />
1 Heart Failure Research Center, Department <strong>of</strong> Anatomy, Embryology and Physiology, Academic Medical<br />
Center, <strong>University</strong> <strong>of</strong> Amsterdam, Amsterdam, The Netherlands. 2 Developmental Biology Institute <strong>of</strong> Marseille-<br />
Luminy, Aix-Marseille <strong>University</strong>, Marseille, France. 3 Department <strong>of</strong> Experimental Biology, <strong>University</strong> <strong>of</strong> Jaén,<br />
Jaén, Spain. 4 Department <strong>of</strong> Genetics and Development, College <strong>of</strong> Physicians and Surgeons <strong>of</strong> Columbia<br />
<strong>University</strong>, Columbia <strong>University</strong> Medical Center, New York, NY, USA<br />
During cardiac development, the anterior second heart field (SHF) contributes cells to the arterial pole and the<br />
posterior SHF to the venous pole. T-box transcription factor Tbx1 is expressed in the SHF and is known to be<br />
involved in development <strong>of</strong> the arterial pole. Here, we aimed to elucidate the early role <strong>of</strong> Tbx1 in the posterior<br />
SHF and derived structures, and its relation to anterior SHF development. Using quantitative 3D reconstruction<br />
methodologies, we found that the proliferative rate and population size <strong>of</strong> SHF cells was severely affected in<br />
Tbx1-deficient mouse embryos. DiI labeling <strong>of</strong> anterior SHF cells in an Fgf10 enhancer trap transgene revealed<br />
that, in the absence <strong>of</strong> Tbx1, the anterior movement <strong>of</strong> anterior SHF cells fails from the 7 somite stage.<br />
Moreover, these anterior SHF cells contribute to the venous pole and differentiate into atrial myocardium by<br />
activating the local atrial gene program. Distal hypoplasia <strong>of</strong> the outflow tract seems to be a late consequence <strong>of</strong><br />
this defect. In addition, the distance between the outflow tract and the inflow tract was greatly reduced and the<br />
subsequent fusion <strong>of</strong> the dorsal mesocardium was consequently delayed. Our findings suggest that Tbx1 is<br />
involved in maintaining the SHF population size from embryonic day (E) 8.0 onward and that Tbx1-deficiency<br />
disrupts proliferation, differentiation and cell fate decisions within the SHF during early heart morphogenesis.<br />
Moreover, despite prepatterning into anterior and posterior subdomains, cardiac progenitor cells <strong>of</strong> the SHF<br />
seem to be naïve in the absence <strong>of</strong> Tbx1.<br />
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S7.6 Retinol Dehydrogenase 10: Roles in Embryo Pharyngeal Patterning and a Model to Understand<br />
How Retinoid Gradients Prevent Congenital Birth Defects<br />
Muriel Rhinn a , Pascal Dollé a , Richard Finnell b , and Karen Niederreither b<br />
a IGBMC, Illkirch, France<br />
b Dept <strong>of</strong> Nutritional Sciences, Dell Pediatric Research Institute, The <strong>University</strong> <strong>of</strong> Texas, Austin.<br />
Retinoic acid (RA), an active vitamin A metabolite, is a key signaling molecule in vertebrate embryos.<br />
Morphogenetic RA gradients are thought to be set up by tissue-specific actions <strong>of</strong> retinaldehyde<br />
dehydrogenases (RALDHs) and catabolizing enzymes (CYP26s) and are important in preventing congenital<br />
malformations. The retinol to retinaldehyde conversion was believed to be achieved by several redundant<br />
enzymes; however, a random mutagenesis screen identified retinol dehydrogenase 10 (RDH10) as responsible<br />
for a homozygous lethal phenotype (Rdh10Trex mutants: Sandell et al., Genes Dev. 21, 1113-1124, 2007) with<br />
typical features <strong>of</strong> RA deficiency. We report the production and characterization <strong>of</strong> novel murine Rdh10 loss <strong>of</strong><br />
function alleles generated by gene targeting. We show that, although the Rdh10-/- mutants die at an earlier<br />
stage than Rdh10Trex mutants, their molecular patterning defects do not reflect a complete state <strong>of</strong> RA<br />
deficiency. Genetic models <strong>of</strong> RA/RALDH2 deficiency p roduces hypoplastic development <strong>of</strong> the 3rd to 6th<br />
branchial arches, a malformation disrupting cardiac outflow tract septation. In Raldh2-/- mutants this defect<br />
cannot be restored by maternally administered RA. In Rdh10-/- mutants, pharyngeal growth and patterning is<br />
restored by administering retinaldehyde to pregnant mothers. We hence obtain viable Rdh10-/- mutants, free <strong>of</strong><br />
lethal congenital malformations. This is the first demonstration <strong>of</strong> rescue <strong>of</strong> an embryonic lethal phenotype by<br />
simple maternal administration <strong>of</strong> the missing retinoid compound. These results underscore the importance <strong>of</strong><br />
maternal retinoids in preventing congenital birth defects, and lead to a revised model on the enzymatic control <strong>of</strong><br />
embryonic RA distribution.<br />
7.7 Arid3b is required for heart development<br />
Veronica Uribe, Claudio Badia-Careaga, Jesus C. Casanova and Juan Jose Sanz-Ezquerro*<br />
Department <strong>of</strong> Cardiovascular Development and Repair, Centro Nacional de Investigaciones Cardiovasculares,<br />
Melchor Fernandez Almagro, 3, 28029 Madrid, Spain<br />
ARID3b is a transcription factor from the highly conserved ARID family, whose members have important roles in<br />
embryonic development and cancer. ARID3b-null mice die during early stages <strong>of</strong> embryonic development<br />
(E10.5) presenting severe defects in the cardiovascular system. However, its roles in development are not fully<br />
understood. Our aim is to address the function <strong>of</strong> Arid3b in the developing heart. We have analysed the<br />
expression pattern <strong>of</strong> this gene during mouse embryo development. Arid3b is expressed from early stages in the<br />
myocardium <strong>of</strong> the tubular hear and also in the cardiac precursors <strong>of</strong> the pharyngeal mesoderm. Later, its<br />
expression gets restricted to the second heart field derived poles <strong>of</strong> the heart. Using Arid3b deficient mice, we<br />
have analyzed the cardiac alterations produced by its absence. Three main defects can be highlighted: a<br />
noticeable shortening <strong>of</strong> the outflow tract; a reduction <strong>of</strong> the size <strong>of</strong> the inflow region with abnormal atria<br />
formation; and an alteration in the development <strong>of</strong> the atrio-ventricular canal (AVC), including defective<br />
formation <strong>of</strong> the AV cushions. Expression <strong>of</strong> several molecular markers <strong>of</strong> both secondary heart field and heart<br />
chambers is altered in mutant embryos. The defects found in mutant embryos and in vitro cell culture data<br />
support a hypothesis that Arid3b could be controlling the addition <strong>of</strong> precursor cells from the second heart field<br />
to the heart by regulating cell motility. Moreover, Arid3b seems to regulate the proper specification and<br />
differentiation <strong>of</strong> the myocardium in heart chambers versus the atrioventricular canal and to be involved in EMT.<br />
7.8 Multiple influences <strong>of</strong> blood flow on cardiomyocyte hypertrophy in the embryonic zebrafish heart<br />
Yi-Fan Lin 1, 2 , Ian Swinburne 2 1, 2<br />
, and Deborah Yelon<br />
1 2<br />
Division <strong>of</strong> Biological Sciences, <strong>University</strong> <strong>of</strong> California, San Diego, La Jolla, CA 92093 USA; Skirball Institute<br />
<strong>of</strong> Biomolecular Medicine, New York <strong>University</strong> School <strong>of</strong> Medicine, New York, NY 10016 USA<br />
Cardiomyocyte hypertrophy is a complex cellular behavior involving coordination <strong>of</strong> cell size expansion and<br />
my<strong>of</strong>ibril content increase. Here, we investigate the contribution <strong>of</strong> cardiomyocyte hypertrophy to cardiac<br />
chamber emergence, the process during which the primitive heart tube transforms into morphologically distinct<br />
chambers and increases its contractile strength. Focusing on the emergence <strong>of</strong> the zebrafish ventricle, we<br />
observed trends toward increased cell surface area and my<strong>of</strong>ibril content. To examine the extent to which these<br />
trends reflect coordinated hypertrophy <strong>of</strong> individual ventricular cardiomyocytes, we developed a method for<br />
tracking cell surface area changes and my<strong>of</strong>ibril dynamics in live embryos. Our data reveal a previously<br />
unappreciated heterogeneity <strong>of</strong> ventricular cardiomyocyte behavior during chamber emergence: although<br />
150
cardiomyocyte hypertrophy was prevalent, many cells did not increase their surface area or my<strong>of</strong>ibril content<br />
during the observed timeframe. Despite the heterogeneity <strong>of</strong> cell behavior, we <strong>of</strong>ten found hypertrophic cells<br />
neighboring each other. Next, we examined the impact <strong>of</strong> blood flow on the regulation <strong>of</strong> cardiomyocyte<br />
behavior during this phase <strong>of</strong> development. When blood flow through the ventricle was reduced, cell surface<br />
area expansion and my<strong>of</strong>ibril content increase were both dampened, and the behavior <strong>of</strong> neighboring cells did<br />
not seem coordinated. Together, our studies suggest a model in which hemodynamic forces have multiple<br />
influences on cardiac chamber emergence: promoting both cardiomyocyte enlargement and my<strong>of</strong>ibril<br />
maturation, enhancing the extent <strong>of</strong> cardiomyocyte hypertrophy, and facilitating the coordination <strong>of</strong> neighboring<br />
cell behaviors.<br />
7.9 An essential role for H3K4 methyltransferase Mll2 in cardiac development<br />
SiangYun Ang 1 , Ji-Eun Lee 2 , Kai Ge 2 , Gregory R. Dressler 3 , Benoit G. Bruneau 1<br />
1 Gladstone Institute <strong>of</strong> Cardiovascular Disease, <strong>University</strong> <strong>of</strong> California, San Francisco.<br />
2 Adipocyte Biology and Gene Regulation Section, National Institute <strong>of</strong> Diabetes and Digestive and Kidney<br />
Diseases, National Institutes <strong>of</strong> Health. 3 Department <strong>of</strong> Pathology, <strong>University</strong> <strong>of</strong> Michigan, Ann Arbor.<br />
During heart development, gene expression is tightly regulated by dynamic chromatin changes. H3K4<br />
methylation is a histone modification that promotes transcriptional activation at associated gene loci. Mutations<br />
in MLL2 (or ALR/ KMT2D), a H3K4 methyltransferase, leads to Kabuki syndrome and is <strong>of</strong>ten associated with<br />
congenital heart defects. Consequently, we hypothesize that H3K4 methylation is required during heart<br />
development, and Mll2 is a key methyltransferase involved in positively regulating cardiac gene expression.<br />
Early embryonic lethality <strong>of</strong> Mll2 Δ/Δ mutants preclude assessment <strong>of</strong> its role in heart development. To determine<br />
the temporal requirement for Mll2 during cardiogenesis, we deleted Mll2 in second heart field (SHF) progenitors<br />
(Isl1::Cre) and in differentiating ventricular myocytes (Nkx2.5::Cre). We observed embryonic lethality and<br />
ventricular septal defects (VSD) in both Isl1::Cre and Nkx2.5::Cre deletion mutants. However, Isl1Cre;Mll2 fl/fl<br />
mutants also exhibited persistent truncus arteriosus (PTA) and thinned myocardium, suggesting a differential<br />
temporal requirement for Mll2 during heart development. Since Mll2 is not known to have intrinsic DNA binding<br />
specificity, we identified a candidate protein Ptip, a subunit unique to the Mll2 methyltransferase complex, which<br />
may recruit Mll2 to target genes. Isl1::Cre;Ptip fl/fl mutants had a similar phenotype to Isl1::Cre;Mll2 fl/fl mutants,<br />
but Nkx2.5::Cre;PTIP fl/fl mutants were normal. These results suggest that Ptip may only be required for Mll2<br />
recruitment at specific developmental stages. Overall, our work demonstrates that H3K4 methylation, as<br />
mediated by Mll2 and Ptip, is important for cardiac development. Future experiments will identify the cell types<br />
responsible for these cardiac defects and define the underlying molecular mechanism.<br />
7.10 Regulation <strong>of</strong> myocardial proliferation in the developing embryo by Gata4 and Tbx5<br />
Chaitali Misra PhD 1 , Sheng Wei Chang BS 1 , Hannah Huang BS 1 , William T. Pu MD 4 , Vidu Garg MD 1,2,3<br />
1 Center for Cardiovascular and Pulmonary Research and the Heart Center, Nationwide Children’s Hospital and<br />
2 Departments <strong>of</strong> Pediatrics and 3 Molecular Genetics, The Ohio State <strong>University</strong>, Columbus, Ohio 43205,<br />
4 Department <strong>of</strong> Cardiology, Children’s Hospital Boston and Harvard Medical School, Boston, MA 02115,<br />
Gata4 and Tbx5 are key transcription factors that play central roles in heart development. Gata4;Tbx5<br />
compound heterozygote mice suffer early lethality and display complete atrioventricular septal defects, thin<br />
myocardium, and decreased myocardial proliferation, but the molecular mechanisms underlying this cardiac<br />
phenotype are unknown. To characterize this genetic interaction, the expression <strong>of</strong> Gata4 and Tbx5 expression<br />
during heart development was examined. Gata4 and Tbx5 are co-expressed in the atria and ventricle until<br />
E13.5, but afterward Tbx5 expression decreases in the ventricle. Consistent with this, thinning <strong>of</strong> the atrial and<br />
ventricular walls in compound heterozygotes is present in E11.5-E13.5 embryos. Gata4 and Tbx5 co-localize in<br />
atria and ventricle until E14.5, and subsequently co-localization is only maintained in the atria. In vivo coimmunoprecipitation<br />
data will be presented to demonstrate this interaction at various developmental timepoints.<br />
To determine the functional significance in vivo, we disrupted the myocardial interaction <strong>of</strong> Gata4 and Tbx5 by<br />
generating mice that were heterozygous for Gata4 in the myocardium and also haploinsufficient for Tbx5<br />
(Gata4 flox/wt ;αMHC-Cre + ;Tbx5 +/- ). These mice recapitulated the cardiac phenotype <strong>of</strong> Gata4;Tbx5 compound<br />
heterozygotes. The expression <strong>of</strong> Cdk2 and Cdk4, two genes important for cell cycle proliferation, was<br />
decreased in Gata4;Tbx5 compound heterozygote hearts. The cardiac phenotype <strong>of</strong> Cdk2 and Cdk4 double<br />
knockout mice is similar to the Gata4;Tbx5 compound heterozygotes, and Cdk4 has previously been<br />
demonstrated to be a direct target <strong>of</strong> Gata4 (Berthet, 2006; Rojas, 2008). Detailed analysis <strong>of</strong> the regulation <strong>of</strong><br />
Cdk2 and Cdk4 by Gata4 and Tbx5 will be presented.<br />
151
7.11 Mef2c Regulates Transcription <strong>of</strong> Cartilage Link Protein 1 in the Developing Heart<br />
Marie M Lockhart 1 , Elaine Wirrig 2 , Aimee Phelps 1 , Russell Norris 1 , Andy Wessels 1<br />
1 Medical <strong>University</strong> <strong>of</strong> South Carolina<br />
2 <strong>University</strong> <strong>of</strong> Cincinnati<br />
Previous studies by the Wessels lab have demonstrated that Hyaluronan and Proteoglycan binding Link Protein<br />
1 (Hapln1 or Crtl1/cartilage link protein) is involved in heart development. Crtl1/Hapln1 is an extracellular matrix<br />
(ECM) protein that stabilizes the interaction between hyaluronan and versican and is expressed in endocardial<br />
and endocardially derived cells in the developing heart, including cells in the atrioventricular (AV) and outflow<br />
tract (OFT) cushions. Hapln1/Crtl1 knockout mice have a range <strong>of</strong> cardiovascular malformations such as thin<br />
myocardium, atrioventricular septal defects (AVSD), and decreased trabeculation. Histological analysis <strong>of</strong><br />
Hapln1/Crtl1 knockout mice reveals there is decreased expression <strong>of</strong> the Crtl1 binding-partners versican and<br />
that reduced expression <strong>of</strong> these ECM proteins may contribute to the cardiovascular malformations observed.<br />
Investigations into the transcriptional regulation <strong>of</strong> the Crtl1 gene have resulted in the finding that the cardiac<br />
transcription factor Mef2c binds to the Crtl1 promoter to regulate its expression in the endocardium and valve<br />
mesenchyme <strong>of</strong> the developing heart.<br />
7.12 Shroom3 deficient mice show congenital heart defects<br />
R.R. Halabi, S. A. Grover and T.A. Drysdale<br />
Children’s Health Research Institute, Department <strong>of</strong> Paediatrics, Physiology and Pharmacology and<br />
Collaborative Graduate Program in Developmental Biology, Western <strong>University</strong>, Canada<br />
Congenital heart defects (CHD) are associated with a number <strong>of</strong> genetic and environmental risk factors and<br />
remain one <strong>of</strong> the most common birth defects - affecting approximately 1-5% <strong>of</strong> newborns. Numerous<br />
transcription factors have been implicated in CHDs, however how their transcriptional activity relates to defects<br />
in morphogenesis remains to be explored. Shroom3 is an actin binding protein found to be essential for neural<br />
tube closure in mouse, Xenopus and chick by its ability to confer apical constriction. Shroom3 has also recently<br />
been associated with heterotaxy in human patients. Whole mount in situ hybridization for Shroom3 has<br />
revealed its expression within the forming heart <strong>of</strong> Xenopus and loss <strong>of</strong> shroom3 activity results in malformed<br />
hearts. Mice homozygous C57BL/6J Shroom3 gene trap insertion die at birth due to severe exencephaly but<br />
there is no information whether there are also cardiac defects. Here we provide evidence <strong>of</strong> multitude CHDs in<br />
the homozygous mutant mice. The homozygotes exhibit both atrial and ventricle septal defects as well as<br />
persistent hypertrophy <strong>of</strong> the ventricles, disorganized trabeculae and immaturity in the valves. Taking<br />
advantage <strong>of</strong> the lacZ gene trap, we have also characterized the expression <strong>of</strong> shroom3 during cardiogenesis.<br />
Our study is an initial step in characterizing the cell activities that drive cardiac morphogenesis and that result in<br />
CHDs when disrupted.<br />
7.13 Function <strong>of</strong> Ddc in fetal and neonatal heart development.<br />
Adam Prickett 1 , Heba Saadeh 1 , Reiner Schulz 1 , H. Scott Baldwin 2 and Rebecca J. Oakey 1<br />
1 King’s College London, Department <strong>of</strong> Medical and Molecular Genetics, 8th Floor Tower Wing, Guy’s Hospital,<br />
London SE1 9RT, United Kingdom; 2 Department <strong>of</strong> Pediatrics, Vanderbilt <strong>University</strong> Medical Center, B3301<br />
MCN, Nashville, Tennessee<br />
Ddc_exon1a is expressed solely from the paternally inherited allele in the developing and neonatal mouse heart,<br />
it is the only gene known to exhibit heart specific imprinting and is expressed from both parental alleles in all<br />
other tissues. Ddc is predominately expressed in the developing myocardium, which points to a role in fetal<br />
heart development. We have utilized knockout (DdcKO) mice, which harbour a germline deletion <strong>of</strong> Ddc, to<br />
examine its role in fetal heart development. Using microarray and episcopic fluorescence image capture<br />
technology (EFIC) we have examined heterozygous Ddc knockouts where the mutated allele has been inherited<br />
from the father for differential gene expression, gross cardiac phenotypes and embryonic growth perturbations.<br />
Our microarray results indicate that Ddc may play a role in cellular proliferation and morphogenesis <strong>of</strong> the<br />
developing myocardium by impacting the expression <strong>of</strong> around 20 genes involved specifically in ventricle<br />
formation. We also present EFIC data illustrating morphological differences between wild type and Ddc<br />
knockout hearts.<br />
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7.14 Roles <strong>of</strong> mesodermally-expressed integrin a5 during cardiovascular development<br />
Dong Liang, Shuan-Yu Hou, Sophie Astr<strong>of</strong><br />
Center for Translational Medicine, Jefferson Medical College, Thomas Jefferson <strong>University</strong><br />
Integrins are heterodimeric cell adhesion receptors. As major mediators <strong>of</strong> cell-ECM, integrins play important<br />
roles in cardiovascular development and other physiological and pathological processes. Integrin a5b1 acts as<br />
one <strong>of</strong> the main fibronectin (FN) receptors during embryogenesis, in part by binding to the RGD region <strong>of</strong> FN.<br />
Integrin a5 -null mouse embryos show very severe defects in cardiovascular development and die by embryonic<br />
day 10.5. Previously, we found that integrin a5 regulates the establishment <strong>of</strong> left-right asymmetry in mice and<br />
fish and plays an essential role in proliferation and survival <strong>of</strong> the cardiac neural crest-derived cells. These<br />
studies were conducted using global integrin a5-null mutants. In order to understand tissue-dependent roles <strong>of</strong><br />
integrin a5 in embryonic patterning and neural crest development, we conditionally ablated integrin a5 in<br />
mesoderm using Mesp1Cre strain <strong>of</strong> mice. Descendants <strong>of</strong> Mesp1Cre-expressing cells give rise to the earliestknown<br />
cardiac progenitors, all endothelial progenitors and entire pharyngeal mesoderm. We found that<br />
conditional ablation <strong>of</strong> integrin a5 using Mesp1Cre leads to embryonic lethality by E16.5 due to severe defects in<br />
the morphogenesis <strong>of</strong> the cardiac outflow vasculature. In addition, majority <strong>of</strong> the mutants have defective<br />
thymus development, and some display aberrant development <strong>of</strong> the right accessory lobe <strong>of</strong> the lung. Defects in<br />
cardiovascular development included hemorrhage, double aortic arch, right sided aorta, left-sided aorta<br />
accompanied by the right sided ductus arteriosus, double outlet right ventricle, interrupted aortic arch,<br />
retroesophageal right subclavian artery, muscular ventricular septum defects and thin compact zone in the<br />
hearts <strong>of</strong> the mutants. Further in situ hybridization and immunohistochemistry studies are being conducted to<br />
determine the mechanisms underlying these morphogenetic defects. These findings will facilitate further<br />
understanding <strong>of</strong> the interactions between mesodermal cells and neural crest cells during cardiovascular<br />
development and the importance <strong>of</strong> integrin signaling in these processes.<br />
7.15 Critical roles <strong>of</strong> Mycn during myocardial wall morphogenesis<br />
Cristina Harmelink and Kai Jiao, MD, PhD.<br />
Department <strong>of</strong> Genetics, UAB, Birmingham, AL.<br />
Our previous study revealed Mycn as a direct target <strong>of</strong> the BMP signaling pathway in developing hearts. The<br />
role <strong>of</strong> BMP signaling in regulating cardiogenesis has been well established. Furthermore, human genetic<br />
studies revealed that haploinsufficiency for MYCN causes Feingold syndrome, a developmental disease<br />
characterized in part by heart defects. In this study we test the hypothesis that myocardial Mycn is essential for<br />
normal myocardial wall morphogenesis through a conditional gene inactivation approach. Loss <strong>of</strong> myocardial<br />
Mycn caused embryonic lethality at midgestation. Mutants displayed severe hypoplastic myocardial walls, which<br />
is caused by both decreased cell proliferation and reduced cell size, but not by increased cell death. Expression<br />
<strong>of</strong> cell cycle regulatory genes was reduced in mutants. Furthermore, Mycn promotes cell growth through<br />
upregulating p70SK expression. Deletion <strong>of</strong> Mycn led to incomplete trabeculation, resembling mouse models<br />
with disruption in the Nrg-1/EphbB pathway. Treating embryonic hearts with an Ephrin-specific blocker severely<br />
reduced Mycn expression, suggesting that Mycn is a key target <strong>of</strong> Ephrin signaling in promoting trabeculation.<br />
Deletion <strong>of</strong> Mycn also eliminated NRG1 and BMP10 upregulated cell proliferation in cultured embryonic hearts,<br />
supporting its essential role in mediating activities <strong>of</strong> BMP and EGF signaling during cardiogenesis. Deletion <strong>of</strong><br />
Mycn did not lead to pre-maturation <strong>of</strong> embryonic cardiomyocytes as evidenced from examining embryonic and<br />
adult specific cardiomyocyte markers. In conclusion, our study reveals Mycn as a key transcription factor<br />
mediating activities <strong>of</strong> multiple signaling pathways in promoting cardiomyocyte proliferation and myocardial wall<br />
morphogenesis.<br />
7.16 Cyp26 enzymes are required during two distinct phases for proper patterning <strong>of</strong> zebrafish heart<br />
chambers<br />
Ariel Rydeen 1,2,3 and Joshua Waxman 2,3<br />
1 Molecular Developmental Biology Graduate Program, <strong>University</strong> <strong>of</strong> Cincinnati College <strong>of</strong> Medicine, 2 The Heart<br />
Institute and Molecular Cardiovascular Biology Division, 3 Cincinnati Children’s Hospital Medical Center,<br />
Cincinnati, OH<br />
Normal heart development requires appropriate levels <strong>of</strong> retinoic acid (RA) signaling, with too much or little<br />
being teratogenic. One way the amount <strong>of</strong> RA is moderated in the embryo is by Cyp26 enzymes, which<br />
metabolize RA into easily degraded derivatives. However, the role Cyp26 enzymes play during heart<br />
development has not yet been addressed. In zebrafish, we found that two Cyp26 enzymes, Cyp26a1 and<br />
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Cyp26c1, are expressed in the anterior lateral plate mesoderm marginally overlapping with the early cardiac<br />
progenitors. While singular knockdown <strong>of</strong> Cyp26a1 or Cyp26c1 does not overtly affect heart development, the<br />
hearts <strong>of</strong> the Cyp26a1 and Cyp26c1 deficient embryos are linearized with smaller ventricles and dilated atria by<br />
48 hours post fertilization (hpf). Interestingly, the phenotype observed in double morphants does not replicate<br />
the teratogenic effects seen when embryos are treated with high concentrations <strong>of</strong> RA, which can eliminate<br />
cardiomyocytes. Closer examination <strong>of</strong> when cardiomyocytes are affected revealed two phases <strong>of</strong> Cyp26<br />
enzyme requirement. We find an earlier role where loss <strong>of</strong> Cyp26 enzymes leads to increased atrial<br />
cardiomyocyte differentiation, which translates into more atrial cells at 48 hpf. Although we do not find an earlier<br />
effect on ventricular cardiomyocyte differentiation, we find ventricle cardiomyocyte number is reduced by 48 hpf<br />
and progressively lost through 72 hpf. Therefore, our results suggest a previously unappreciated model where<br />
Cyp26 enzymes are required to prevent increases in RA signaling at two distinct phases to allow for proper<br />
heart development.<br />
7.17 Development <strong>of</strong> atrial septum: Chick vs Mouse<br />
Moreno-Rodriguez RA, Reyes L, Krug E, H<strong>of</strong>fman S and Markwald RR.<br />
Regenerative Medicine Cardiovascular Biology Center. MUSC. Charleston, SC, USA.<br />
Six different cell populations participate in atrial septation: the atrial mesenchymal cap (AMC); dorsal<br />
mesenchymal protrusion (DMP); mesenchyme from the superior and inferior cushions <strong>of</strong> the AV canal; a<br />
myocardial cell population from the septum primum (SI); and from the septum secundum (SII). In placental<br />
mammals those cell populations are integrated during the three phases <strong>of</strong> atrial septation: Phase I, is initiated by<br />
the formation <strong>of</strong> the AMC, which together with the inferior cushion, superior cushion and by the DMP, encircled<br />
the ostium primum. Over the SI openings are formed that coalescent to form the ostium secundum; and the<br />
ostium primum is closed by the fusion <strong>of</strong> the AMC with the inferior cushion. Phase II, includes the formation <strong>of</strong><br />
the SII which grows ventrally until it forms a semilunar-shaped structure (Foramen Ovale). Phase III, the SI and<br />
SII fuse to form the definitive atrial septum. It is important to mention that avian and lower mammals only<br />
present a Phase I. The Periostin expression correlates with the transformation <strong>of</strong> the muscular SI into<br />
mesenchyme, and its expression is maintained until the closure <strong>of</strong> ostium II. We report on the development <strong>of</strong><br />
chick culture model to study the molecular mechanisms (periostin initially) that govern the Phase I <strong>of</strong> atrial<br />
septation that is extendable to the mouse. Supported by: 2R01HL033756-27; Leducq (07CVD04); COBRE (P20<br />
RR016434/P20 RR021949-01A2).<br />
7.18 IGF2 Signaling Regulates Cardiomyocyte Proliferation<br />
Hua Shen 1 , Susana Cavallero 1 , Peng Li 1 , Thomas Brade 2 , Gregg Duester 2 , Miguel Constancia 3 and Henry<br />
Sucov 1<br />
1 <strong>University</strong> <strong>of</strong> Southern California Keck School <strong>of</strong> Medicine, Broad Center for Regenerative Medicine and Stem<br />
Cell Research, 2250 Alcazar Street, CSC240, Los Angeles, CA 90033, USA. 2 Sanford-Burnham Medical<br />
Research Institute, Development and Aging Program, 10901 North Torrey Pines Road, La Jolla, CA 92037,<br />
USA. 3 Laboratory <strong>of</strong> Developmental Genetics and Imprinting, The Babraham Institute, Babraham Research<br />
Campus, Cambridge, UK<br />
Our recent results show that IGF2 is the major epicardial mitogen that supports embryonic ventricular<br />
cardiomyocyte proliferation. For this analysis, we employed cardiac-specific (Nkx2.5Cre) IGF receptor mutants<br />
and global Igf2 ligand mutant embryos. We have now crossed Nkx2.5Cre with a conditional Igf2 allele and<br />
confirmed that the source <strong>of</strong> the IGF2 ligand is within the heart (within the Nkx2.5Cre recombination domain).<br />
We are now in the process <strong>of</strong> crossing the conditional Igf2 allele with epicardial-specific as well as endocardial-<br />
and myocardial-specific Cre lines to further define the source <strong>of</strong> this ligand. Embryos globally lacking the retinoic<br />
acid (RA) receptor RXRa are also compromised in cardiomyocyte proliferation and heart development.<br />
Epicardial Igf2 expression is diminished in RXRa mutants, linking these two genes in a common pathway;<br />
however, the Igf2 gene is not directly activated by RA. We crossed a number <strong>of</strong> Cre lines and found<br />
mesodermal-specific Mesp1Cre o r neural crest cell lineage-specific Wnt1Cre did not replicate the phenotype <strong>of</strong><br />
global RXRa deficiency. However, the more broadly active mesodermal Brachyury (T)-Cre impacted heart<br />
development. We had previously shown that RA acts in the fetal liver to directly control expression <strong>of</strong> the<br />
cytokine erythropoietin (EPO). T-Cre is active in the fetal liver, whereas Mesp1Cre is inefficient in liver<br />
recombination. We found that EPO addition rescued Igf2 expression in RA-deficient embryonic hearts. Our<br />
results support a model in which RA signaling in mesodermal cells <strong>of</strong> the fetal liver supports Epo expression,<br />
which then influences epicardial Igf2 expression and subsequent cardiomyocyte proliferation.<br />
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7.19 Roles <strong>of</strong> LRRC10, a cardiac specific factor in heart development and function<br />
Matt J. Brody 1 , Timothy A. Hacker 2 , Sergei Tevosian 3 , and Youngsook Lee 1<br />
1 Department <strong>of</strong> Cell and Regenerative Biology, Molecular Environmental Toxicology Center, 2 Department <strong>of</strong><br />
Medicine, <strong>University</strong> <strong>of</strong> Wisconsin-Madison; 3 Department <strong>of</strong> Genetics, Dartmouth Medical School, Hanover, NH<br />
Leucine Rich Repeat Containing protein 10 (LRRC10) has been identified as a cardiac-specific factor.<br />
Knockdown <strong>of</strong> lrrc10 in zebrafish embryos caused severe cardiac morphogenic defects including a cardiac<br />
looping failure and a decrease in cardiac output, resulting in embryonic lethality. Since the roles <strong>of</strong> LRRC10 in<br />
mammalian hearts remain to be elucidated, Lrrc10 knock out (KO) mice were investigated. Lrrc10 KO mice<br />
exhibit systolic dysfunction in utero and develop dilated cardiomyopathy in early postnatal life. LRRC10 is<br />
detected both in cytoplasm and nuclei <strong>of</strong> embryonic cardiomyocytes but shows striations in adult<br />
cardiomyocytes. Gene expression pr<strong>of</strong>iling experiments identified multiple defective molecular pathways both in<br />
embryonic and adult Lrrc10 KO hearts, but revealed distinct molecular defects in pre vs post natal hearts.<br />
Pathway analysis identified regulation <strong>of</strong> the actin cytoskeleton as a significantly upregulated pathway in<br />
embryonic Lrrc10 KO hearts and signal transduction pathways involving AKT and PKCε are altered in adult<br />
Lrrc10 KO hearts. Yeast two-hybrid screening and coimmununoprecipitation studies indicate that LRRC10<br />
interacts with α -actinin and actin is<strong>of</strong>orms, implying that LRRC10 serves as a mechanical link between the<br />
my<strong>of</strong>ilament Z-disc and the cytoskeleton. Our study demonstrates that the developmental perturbation <strong>of</strong><br />
cardiac biology in the absence <strong>of</strong> LRRC10 results in adult heart disease. The precise molecular function <strong>of</strong><br />
LRRC10 is being investigated, which will enhance our understanding <strong>of</strong> congenital heart defects and heart<br />
disease. In summary, we identify LRRC10 as a gene crucial for embryonic cardiac function and proper<br />
development and a novel dilated cardiomyopathy candidate gene.<br />
7.20 Signalling via Bmp Type I receptor Alk2 (AcvrI) in second heart field cells and/or endocardium is<br />
required for right ventricle and outflow tract development<br />
Penny S. Thomas and Vesa Kaartinen<br />
Biologic and Materials Sciences, <strong>University</strong> <strong>of</strong> Michigan, Ann Arbor<br />
Bmps are required for normal development <strong>of</strong> right ventricle, outflow tract and great vessels. Using the MEF2c-<br />
AHD-Cre driver, we are investigating the role <strong>of</strong> Bmp signaling through Alk2. Post-septation conditional Alk2-null<br />
embryos show a range <strong>of</strong> OFT/truncal phenotypes, from double outlet right ventricle to common arterial trunk,<br />
and abnormal embryonic RV morphology is recognizable from E9. A number <strong>of</strong> studies have suggested that the<br />
balance between Bmp and Fgf signaling, and Bmp-induced miRNAs, play key roles in this process. Using Q-<br />
RTPCR, we examined expression <strong>of</strong> key genes in RV+OFT at E9 and, using R26R-YFP and FACS sorting, in<br />
recombined SHF cells outside the heart at E9 and at E10. Expression <strong>of</strong> Bmp signaling target Smad6 was<br />
reduced in mutants in all three tissues. Although Isl1 was increased in RV+OFT in Alk2 mutant RV+OFTs, Fgf10<br />
showed no consistent difference. Tbx1 expression was inconsistently elevated in E9 SHF, but Isl1 and Fgf10<br />
were not different from controls. Levels <strong>of</strong> miRNA17 and 20a did not differ between Alk2 mutants and controls in<br />
either dissected E9 SHF-enriched tissues or E9 hearts. These results suggest that the altered expression <strong>of</strong><br />
genes regulated by Bmp signaling happen at different times/in different populations <strong>of</strong> cells, and that miRNA17<br />
and 20a do not play a major role in our Alk2 cko model. We have detected Alk2 in SHF cells by WMT ISH and<br />
using an Alk2 reporter BAC line at E8, showing that MEF2c-AHD-Cre recombination in these cells could directly<br />
underly at least the earliest abnormal phenotype.<br />
7.21 WNT/β-catenin Signaling Promotes Growth <strong>of</strong> Ventricular Myocytes from the First- and Second-<br />
Heart-Field<br />
J.W. Buikema MD/PhD Student 1, 2, 3 , J.P.G. Sluijter PhD 2 , P.A.F.M. Doevendans, MD/PhD 2 , I.J. Domian,<br />
MD/PhD<br />
1, 3, 4<br />
1 Cardiovascular Research Center, Massachusetts General Hospital, Boston, MA 02114, USA. 2 Department <strong>of</strong><br />
Cardiology, Division <strong>of</strong> Heart & Lungs, <strong>University</strong> Medical Center Utrecht, 3584 CX Utrecht, The Netherlands.<br />
3 Harvard Medical School, Boston, MA 02115, USA. 4 Harvard Stem Cell Institute, Cambridge, MA 02138, USA<br />
Elucidating the pathways that control ventricular growth and development is key to developing novel approaches<br />
in regenerative cardiovascular therapy. Cells from the first- and second-heart-field give rise to distinct regions <strong>of</strong><br />
the four-chambered heart. Previously it is shown that canonical WNT signaling has bimodal effects on<br />
differentiation and growth <strong>of</strong> the developing heart fields (E7.0-9.5) and in the early heart (E9.5-12.5). However it<br />
remains unclear what the role <strong>of</strong> WNT signaling is in later stages <strong>of</strong> cardiac development. We asked the specific<br />
question <strong>of</strong> what role canonical WNT signaling plays in late ventricular development. Therefore we used an in<br />
155
vitro double transgenic mouse reporter system to isolate ventricular progenitors <strong>of</strong> the first- and second-heartfield.<br />
In these, we overexpressed WNT/β-catenin signaling by a small molecule known to target this pathway.<br />
Upon overregulation <strong>of</strong> WNT we found a significantly higher proportion <strong>of</strong> proliferating ventricular progenitors<br />
and myocytes in both populations. We also looked at the function <strong>of</strong> canonical WNT signaling in controlling heart<br />
growth in vivo. Therefore we stabilized β -catenin in ventricular MYL2 expressing cells using a Cre-LoxP<br />
recombination. At P1 we found an increase in proliferation <strong>of</strong> left and right ventricular myocytes. Preliminary<br />
data shows that P35 mice with MYL2 WNT overexpression have a modest increase in heart size. Altogether<br />
these data suggest that WNT/β-catenin signaling is important in increasing late embryonic and postnatal<br />
ventricular myocyte growth and thereby heart size control.<br />
7.22 BMP Signaling and Atrioventricular Septation<br />
Briggs Laura 1 , Aimee Phelps 1 , Jayant Kakarla 2 , Maurice van den H<strong>of</strong>f 3 , Andy Wessels 1<br />
1 Medical <strong>University</strong> <strong>of</strong> South Carolina; 2 Newcastle <strong>University</strong>; 3 <strong>University</strong> <strong>of</strong> Amsterdam<br />
Atrioventricular septal defects (AVSDs) encompass a spectrum <strong>of</strong> malformations in which deficient septation<br />
permits the chambers <strong>of</strong> the heart to communicate. These defects are relatively common; they comprise 3-5%<br />
<strong>of</strong> all congenital cardiac malformations and are present in approximately 25% <strong>of</strong> children with Down syndrome.<br />
For many years, AVSDs were thought to arise solely from malformation <strong>of</strong> the endocardial cushions. More<br />
recently, however, defective development <strong>of</strong> the dorsal mesenchymal protrusion (DMP), a derivative <strong>of</strong> the<br />
Second Heart Field, has also been implicated in the pathogenesis <strong>of</strong> AVSDs. Insight into the mechanism(s)<br />
governing proper development <strong>of</strong> the DMP is slowly emerging. In this study we present data demonstrating that<br />
BMP signaling is critical for DMP formation and/or maturation. We show that pSMAD1/5/8 is expressed at high<br />
levels in the DMP precursor population, that BMP ligands are expressed around the developing DMP, and that<br />
deletion <strong>of</strong> the BMP type I receptor ALK3 from the DMP precursors results in a hypoplastic DMP and a fully<br />
penetrant AVSD phenotype. While the importance <strong>of</strong> BMPs in the development <strong>of</strong> the outflow tract and AV<br />
junction is well established, our work indicates a novel role for BMP signaling at the venous pole <strong>of</strong> the heart.<br />
7.23 Hedgehog-dependent atrial septum progenitors are required for cardiac septation<br />
Andrew D. H<strong>of</strong>fmann 1 , Holly Yang 2 , Junghun Kweon 2 , Joshua Bosman 2 , Anne M. Moon 3 and Ivan P. Moskowitz 2<br />
1 Committee on Development, Regeneration and Stem Cell Biology, The <strong>University</strong> <strong>of</strong> <strong>Chicago</strong>, <strong>Chicago</strong> IL,<br />
60637, USA. 2 Departments <strong>of</strong> Pediatrics and Pathology, The <strong>University</strong> <strong>of</strong> <strong>Chicago</strong>, <strong>Chicago</strong>, IL, 60637, USA.<br />
3 Department <strong>of</strong> Pediatrics, <strong>University</strong> <strong>of</strong> Utah, Salt Lake City, UT, 84158, USA<br />
Atrial septation is a critical step in separating the systemic and pulmonary circulations in tetrapods and atrial<br />
septal defects are among the most common forms <strong>of</strong> human congenital heart disease. We have previously<br />
demonstrated that the atrial septum is generated by second heart field (SHF) progenitors that receive Hedgehog<br />
(Hh) signaling (H<strong>of</strong>fmann et al., 2009). To identify Hh-dependent pathways for atrial septation, we performed<br />
transcriptional pr<strong>of</strong>iling on SHF tissues <strong>of</strong> wild-type and shh knockout mice. We have identified and verified<br />
genes whose SHF expression is Hh- dependent by RT-PCR and examined several genes by in-situ<br />
hybridization. Interestingly, the expression <strong>of</strong> Hh-dependent transcripts cluster into distinct SHF domains: (1)<br />
genes implicated in proliferation in other contexts, such as FGF8, are expressed in an arch-shaped pattern<br />
lateral to and surrounding the dorsal mesocardium and (2) genes implicated in differentiation in other contexts,<br />
such as BMP4, appear to be located more medial and directly adjacent to the dorsal mesocardium. We have<br />
directly examined the requirement for these pathways in atrial septation and find that removal <strong>of</strong> FGF8, FGFR1<br />
and 2, or BMP4 from SHF tissues results in failure <strong>of</strong> atrial septation. We are currently testing the domain<br />
hypothesis that laterally-expressed genes control posterior SHF proliferation, while medially-expressed genes<br />
trigger differentiation. These results form the basis for investigating the cellular and molecular mechanisms<br />
required for generation <strong>of</strong> the atrial septum from SHF cardiac progenitors.<br />
7.24 THE PLANAR CELL POLARITY SIGNALING PATHWAY IS REQUIRED IN THE SECOND HEART FIELD LINEAGE FOR<br />
OUTFLOW TRACT MORPHOGENESIS<br />
Tanvi Sinha, Bing Wang, Jianbo Wang<br />
Department <strong>of</strong> Cell Biology, <strong>University</strong> <strong>of</strong> Alabama at Birmingham, Birmingham, Alabama-35294<br />
Outflow tract (OFT) malformations underlie a majority <strong>of</strong> human congenital heart defects. The OFT arises in part<br />
from the Second Heart Field (SHF) progenitors in the pharyngeal and splanchnic mesoderm (SpM), outside <strong>of</strong><br />
the initial heart. While SHF proliferation and differentiation have been extensively studied; how these progenitors<br />
are deployed to the OFT remains unclear. Using a set <strong>of</strong> mouse Dishevelled2 (Dvl2) alleles, we demonstrate<br />
156
that the planar cell polarity (PCP) pathway, a branch <strong>of</strong> the β-catenin independent non-canonical Wnt pathway<br />
that regulates cellular polarity and polarized cell behavior during tissue morphogenesis, is required specifically in<br />
the SHF for early OFT morphogenesis and may play a key role in SHF deployment. We find that mutations in<br />
mouse core PCP genes Dvl1/2 and Vangl2 as well as non-canonical Wnt gene Wnt5a result in aberrant cardiac<br />
looping. The looping defect is correlated with severe OFT shortening at embryonic day 9.5, characteristic <strong>of</strong><br />
comprised contribution from the SHF. Consistent with our genetic interaction studies suggesting that Wnt5a<br />
signals through the PCP pathway, Dvl1/2 and Wnt5a mutants display aberrant cell packing along with<br />
diminished actin polymerization and filopodia formation in SHF progenitors in the caudal SpM, where Wnt5a and<br />
Dvl2 are co-expressed specifically. Based on these results, we propose a novel model in which a Wnt5a�Dvl<br />
PCP signaling cascade promotes SHF deployment by promoting actin polymerization and protrusive cell<br />
behavior to continuously recruit SHF progenitors into a cohesive sheet in the caudal SpM, thereby pushing the<br />
SpM rostrally into the OFT.<br />
7.25 Casz1 is Required for Cardiac Development<br />
Kerry Dorr 1,3 , Hanna Labiner 1,3 , Frank L. Conlon 1,2,3<br />
McAllister Heart Institute, Department <strong>of</strong> Biology, Department <strong>of</strong> Genetics<br />
<strong>University</strong> <strong>of</strong> North Carolina, Chapel Hill, NC, USA<br />
Understanding the molecular mechanisms <strong>of</strong> cardiomyocyte cell fate decisions is critical for uncovering<br />
pathologies and treatments for congenital heart disease. The program <strong>of</strong> differentiation and the identity <strong>of</strong> the<br />
molecules and signals that regulate the fate <strong>of</strong> cardiac progenitor cells and their ability to differentiate into the<br />
major functional cell lineages <strong>of</strong> the heart remain important areas <strong>of</strong> study. Here we establish that the zinc finger<br />
transcription factor Castor (Casz1) displays an evolutionarily conserved expression pattern in cardiac tissue. We<br />
have gone on to generate a conditional allele <strong>of</strong> Casz1 and demonstrate that the cardiac-specific ablation <strong>of</strong><br />
Casz1 leads to embryonic lethality with homozygous null mice exhibiting a range <strong>of</strong> cardiac malformations<br />
including ventricular septal defects. To extend and complement these studies, we sought to determine the fate<br />
<strong>of</strong> Casz1 cells in the developing embryo. To this end, we have generated lines <strong>of</strong> mice in which we have<br />
knocked-in an inducible CreERT cassette into the Casz1 locus (Casz1KI-CreERT2). Results <strong>of</strong> fate mapping<br />
studies using the Casz1KI-CreERT2 allele demonstrate that Casz1-expressing cells give rise to cardiac<br />
structures including the atria and interventricular septum. Collectively, these studies demonstrate that Casz1 is<br />
essential for cardiac development and has an essential role in septum formation.<br />
Section 8: Valve Development<br />
S8.1 Computational modeling <strong>of</strong> endocardial cell activation during AV valve formation.<br />
Lagendijk 1 , A.K., Szabo 2 , A., Merks 2 , R. and Bakkers J. 1<br />
1 Hubrecht Institute and <strong>University</strong> Medical Center Utrecht, the Netherlands<br />
2 Netherlands Institute for Systems Biology, Amsterdam, the Netherlands<br />
The development <strong>of</strong> valve structures occurs in distinguishable phases, which are highly conserved. The first<br />
event during valve development is the induction <strong>of</strong> endocardial cushions (ECs) within the atrioventricular (AV)<br />
canal and outflow tract <strong>of</strong> the primitive heart tube. It has been well established that a BMP signal from the<br />
myocardium initiates this event by the induction <strong>of</strong> TGF-β, Has2 (hyaluronic acid synthase 2), and Notch1, as<br />
well as the transcription factors Snail1 and Twist1. The importance <strong>of</strong> regulating Has2 expression in the<br />
endocardium has been exemplified by the observation that in Has2 deficient mice or zebrafish the cardiac jelly<br />
does not expand and ECs fail to form. We recently showed that zebrafish dicer mutant embryos, lacking mature<br />
miRNAs, form excessive endocardial cushions (Lagendijk et al. Circ Res. 2011). By functional screening we<br />
found that miR-23 is both necessary and sufficient for restricting the number <strong>of</strong> activated endocardial cells in the<br />
ECs. In addition, in mouse endothelial cells, miR-23 inhibited a TGF-β–induced endothelial-to-mesenchymal<br />
transition. By in silico screening combined with in vivo testing, we identified Has2, Icat, and Tmem2 as novel<br />
direct targets <strong>of</strong> miR-23. Surprisingly, miR-23 expression is confined to the ECs where its activity is required to<br />
repress Has2. To understand the significance <strong>of</strong> the miR-23-Has2 interaction we applied computational<br />
modeling using a cellular Potts algorithm. The computational model predicts a regulatory network <strong>of</strong> both<br />
positive and negative feedback mechanisms to restrict the activation <strong>of</strong> the endocardial cells to the AV canal.<br />
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S8.2 Tie1 is Required for Semilunar Valve Form and Function<br />
Chris Brown 1 , Xianghu Qu 1 , Kate Violette 1,2 , M-K Sewell 3 , W. David Merryman 3 , Bin Zhou, 4 and H. Scott<br />
Baldwin 1,2<br />
1 Div. <strong>of</strong> Ped. Cardiology. 2 Depts. <strong>of</strong> Cell and Dev. Biol. and 3 Biomedical Engineering, Vanderbilt <strong>University</strong>,<br />
Nashville, TN 4 Genetics, Albert Einstein College <strong>of</strong> Medicine, NY.<br />
The mechanisms regulating late gestational and early postnatal semilunar valve remodeling and maturation are<br />
poorly understood. Tie1 is a receptor tyrosine kinase with broad expression in embryonic endothelium. During<br />
semilunar valve development, Tie1 expression becomes restricted to the turbulent, arterial surfaces <strong>of</strong> the<br />
valves in the perinatal period. Previous studies in our laboratory have demonstrated that Tie1 can regulate<br />
cellular responses to blood flow and shear stress. We hypothesized that Tie1 signaling would regulate the flow<br />
dependent remodeling <strong>of</strong> the semilunar valves associated with the conversion from maternal/placental to<br />
independent neonatal circulation. To circumvent the embryonic lethality, we developed a floxed Tie1 allele and<br />
crossed it to an Nfatc1 P2-Cre line that mediates gene excision exclusively in the endocardial cushion<br />
endothelium. Excision <strong>of</strong> Tie1 resulted in aortic valve leaflets displaying hypertrophy with perturbed matrix<br />
deposition and re modeling without alteration in cell number, proliferation, or apoptosis. Differences were only<br />
detected after birth, increased with age, and were restricted to the aortic valve. RNA sequence analysis<br />
suggests that mutant aortic valves are similar to wildtype pulmonary valves in transcriptional pr<strong>of</strong>ile. The aortic<br />
valves demonstrated insufficiency and stenosis by ultrasound and atomic force microcopy documented<br />
decreased stiffness in the mutant aortic valve consistent with an increased glycosaminoglycan to collagen ratio.<br />
These data suggest that active endocardial to mesenchymal signaling, at least partially mediated by Tie1, is<br />
uniquely required for normal remodeling <strong>of</strong> the aortic but not pulmonary valve in the late gestation and post-natal<br />
animal. Suppt. by RL1HL0952551 (HSB), HL094707 (WDM), HL078881 (BZ).<br />
S8.3 In Vivo Reduction Of Smad2 Concomitant With Proteoglycan Accumulation Results In High<br />
Penetrance Of Bicuspid Aortic Valves<br />
1 Loren Dupuis, 2 Robert B. Hinton and 1 Christine B. Kern<br />
1 Department <strong>of</strong> Regenerative Medicine and Cell Biology, Medical <strong>University</strong> <strong>of</strong> South Carolina, Charleston, SC<br />
29464, 2 Division <strong>of</strong> Cardiology, the Heart Institute, Cincinnati Children's Hospital Medical Center, Cincinnati, OH<br />
45229<br />
Bicuspid aortic valve (BAV) is the most prevalent cardiovascular malformation resulting in two rather than three<br />
mature cusps and occurs in at least 1-2% <strong>of</strong> the population. However, only NOTCH-1 loss <strong>of</strong> function has been<br />
linked to human BAV disease, and there are limited genetically modified mouse models that display BAV. We<br />
reported that loss <strong>of</strong> proteolytic cleavage <strong>of</strong> versican, a proteoglycan abundant in cardiac outlet cushions, results<br />
in myxomatous valves in ADAMTS5 deficient mice. Recently we discovered that a failure to cleave versican was<br />
concomitant with a reduction <strong>of</strong> cell-cell condensation, phosphorylated Smad2 and fibrous ECM organization in<br />
Adamts5 -/- valves. To test the hypothesis that that versican cleavage via ADAMTS5 is required to reduce the<br />
versican-hyaluronan pericellular matrix to elicit Smad2 phosphorylation we further reduced Smad2 in Adamts5 -/-<br />
mice through intergenetic cross. The resulting Adamts5 -/- ;Smad2 +/- mice developed a more dramatic valve<br />
phenotype than Adamts5 -/-; Smad2 +/+ mice and displayed a high penetrance <strong>of</strong> BAV (6/9). Interestingly, the<br />
pulmonary valve was also bicuspid in 5/9 (4/9 displayed both bicuspid PV and BAV). All Adamts5 -/- ;Smad2 +/-<br />
semilunar valves displayed wide hinge regions at the juncture <strong>of</strong> the annulus concomitant with loss <strong>of</strong> fibrous<br />
ECM. These data suggest that the dramatic changes in proteoglycan turnover, during remodeling <strong>of</strong> the truncal<br />
cushions, elicit changes in cell behavior and signaling required for normal semilunar cusp formation. Further<br />
studies <strong>of</strong> the Adamts5 -/- ;Smad2 +/- mice may elucidate a novel etiology <strong>of</strong> BAV pathogenesis and lead to new<br />
pharmacological treatments for valve disease.<br />
S8.4 Filamin-A Regulates Tissue Remodeling <strong>of</strong> Developing Cardiac Valves via a Novel Serotonin<br />
Pathway<br />
Kimberly Sauls 1 , Annemarieke de Vlaming 1 , Katherine Williams 1 , Andy Wessels 1 , Robert A. Levine 2 , Susan A<br />
Slaugenhaupt 3 , Roger R. Markwald 1 , Russell A. Norris 1*<br />
1 Department <strong>of</strong> Regenerative Medicine and Cell Biology, School <strong>of</strong> Medicine, Cardiovascular Developmental Biology Center,<br />
Children’s Research Institute, Medical <strong>University</strong> <strong>of</strong> South Carolina, Charleston, SC, 29425, USA. 2 Noninvasive Cardiac<br />
Laboratory, Department <strong>of</strong> Medicine, Massachusetts General Hospital, Boston, Massachusetts, 02114, USA. 3 Center for<br />
Human Genetic Research, Massachusetts General Hospital/Harvard Medical School, Boston, Massachusetts, 02114, USA<br />
Linkage and sequencing studies <strong>of</strong> patients with myxomatous valvular dystrophy revealed causal mutations in<br />
the Filamin-A gene. These studies defined Filamin-A as integral to valve structure and function. However, the<br />
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mechanisms by which Filamin-A regulates valve cell biology are unknown. Herein we demonstrate that Filamin-<br />
A conditional KO (cKO) mice exhibit enlarged leaflets commencing during fetal valve maturation. This defect is<br />
caused by an inability for valve fibroblasts to efficiently remodel their extracellular matrix environment. Using the<br />
patient mutations as a guide, we identified a novel mechanism by which cooperative interaction between<br />
intracellular serotonin, transglutaminase-2 (TG2), and Filamin-A are required for promoting cytoskeletal-driven<br />
matrix remodeling events. Co-expression <strong>of</strong> serotonin, serotonin transporter (SERT), TG2, and Filamin-A is<br />
restricted to fetal valve development during active matrix remodeling. At this timepoint, immunoprecipitation<br />
experiments demonstrate that serotonin is covalently bound to Filamin-A in vivo and in vitro and is dependent on<br />
intact TG2 activity. Pharmacological and genetic perturbations <strong>of</strong> this interaction demonstrate serotonin<br />
transport inhibition, abrogation <strong>of</strong> TG2 activity, and/or genetic removal <strong>of</strong> Filamin-A result in loss <strong>of</strong> serotonin-<br />
Filamin-A interaction and impaired matrix remodeling. These findings illustrate a novel mechanism by which<br />
intracellular serotonin, TG2 and Filamin-A cooperatively regulate cytoskeletal-driven matrix remodeling. As<br />
such, these data provide mechanistic insight into normal processes driving cardiac valve maturation with added<br />
potential for providing insight into the developmental basis for human degenerative cardiac valve disease.<br />
S8.5 Epicardially-derived Fibroblasts Preferentially Contribute to the Parietal Leaflets <strong>of</strong> the<br />
Atrioventricular Valves in the Murine Heart<br />
Andy Wessels a , Maurice J. B. van den H<strong>of</strong>f b , Richard F. Adamo c , Aimee L. Phelps a Marie M. Lockhart a , Kimberly<br />
Sauls a , Laura E. Briggs a , Russell A. Norris a , Bram van Wijk b , Jose M. Perez-Pomares d , Robert W. Dettman e ,<br />
and John B. E. Burch f<br />
a Medical <strong>University</strong> <strong>of</strong> South Carolina, Charleston, South Carolina, USA. b Academic Medical Center,<br />
Amsterdam, The Netherlands. c The Children’s Hospital <strong>of</strong> Philadelphia, Philadelphia, Pennsylvania, USA.<br />
d <strong>University</strong> <strong>of</strong> Malaga, Spain. e Northwestern <strong>University</strong>, Feinberg School <strong>of</strong> Medicine, <strong>Chicago</strong>, Illinois, USA.<br />
f Fox Chase Cancer Center, Philadelphia, Pennsylvania, USA<br />
The importance <strong>of</strong> the epicardium for valvuloseptal development has been well established. To determine<br />
whether and if so how epicardially derived cells contribute to the developing valves in the murine heart we used<br />
a mWt1/IRES/GFP-Cre mouse to trace the fate <strong>of</strong> EPDCs from embryonic day (ED)10 until 4 months <strong>of</strong> age.<br />
Migration <strong>of</strong> EPDCs into the atrioventricular cushion mesenchyme starts around ED12. As development<br />
progresses, the number <strong>of</strong> EPDCs in the cushions increases significantly, specifically in the leaflets which derive<br />
from the lateral atrioventricular cushions. In these leaflets, the epicardially-derived fibroblasts eventually largely<br />
replace the endocardially-derived cells. Importantly, the contribution <strong>of</strong> EPDCs to the leaflets derived from the<br />
major AV cushions is very limited. The differential contribution <strong>of</strong> EPDCs to the respective leaflets <strong>of</strong> the<br />
atrioventricular valves provides a new paradigm in valve development and could lead to new insights into the<br />
pathogenesis <strong>of</strong> abnormalities that preferentially affect individual components <strong>of</strong> this region <strong>of</strong> the heart. The<br />
notion that there is a significant difference in the contribution <strong>of</strong> epicardially and endocardially derived cells to<br />
the individual leaflets <strong>of</strong> the atrioventricular valves has also important pragmatic consequences for the use <strong>of</strong><br />
endocardial and epicardial cre-mouse models in heart development.<br />
S8.6 Endothelial nitric oxide signaling regulates Notch1 in aortic valve development and disease<br />
Kevin Bosse PhD, Chetan P. Hans PhD, Ning Zhao MS, Sara N. Koenig BA, Nianyuan Huang BS, Anuradha<br />
Guggilam PhD, Ge Tao PhD, Pamela A. Lucchesi PhD, Joy Lincoln PhD, Brenda Lilly PhD and Vidu Garg MD<br />
The Research Institute at Nationwide Children's Hospital<br />
Aortic valve calcification is the third leading cause <strong>of</strong> heart disease in adults and is frequently associated with<br />
bicuspid aortic valve, the most common type <strong>of</strong> cardiac malformation. NOTCH1 and the nitric oxide (NO)<br />
signaling pathway have been implicated in aortic valve development and calcification. Using an established<br />
porcine aortic valve interstitial cell (PAVIC) culture system known to spontaneously calcify, we demonstrate that<br />
gain or loss <strong>of</strong> NO, secreted by endothelial cells, prevents or accelerates calcification <strong>of</strong> PAVICs, respectively.<br />
Overexpression <strong>of</strong> Notch1 prevented the calcification that occurred with inhibition <strong>of</strong> NO, demonstrating the<br />
effects <strong>of</strong> NO on calcification are mediated by Notch1. Furthermore, endothelial cells or addition <strong>of</strong> NO<br />
regulates the nuclear localization <strong>of</strong> Notch1 and affects the expression <strong>of</strong> Hey1, a Notch1 target gene in<br />
PAVICs. To determine if Notch1 and endothelial nitric oxide (eNOS) genetically interact in vivo, we generated<br />
eNOS -/- ;Notch1 +/- compound mutant mice. Embryonic examination revealed cardiac abnormalities in a subset <strong>of</strong><br />
eNOS -/- ;Notch1 +/- mice, which suffered ~70% lethality by postnatal day 10. Surviving compound mutant mice at<br />
8 weeks <strong>of</strong> age demonstrated severely malformed aortic valves. eNOS -/- ;Notch1 +/- mice maintained on high fat<br />
diet for 12 weeks displayed aortic regurgitation and stenosis, thickened aortic cusps and development <strong>of</strong> calcific<br />
nodules on the aortic valve leaflets as compared to age-matched eNOS -/- mice. Overall, these data<br />
159
demonstrate a novel molecular pathway by which endothelial nitric oxide regulates Notch1 signaling in a manner<br />
that is critical for aortic valve development and disease.<br />
8.7 Analysis <strong>of</strong> TGFβ3 function in valve remodeling and aortic valve calcification<br />
Alejandra C Encinas, Robert Hinton # , and Mohamad Azhar<br />
Program in Developmental Biology and Neonatal Medicine, Herman B Wells Center for Pediatric Research,<br />
Indiana <strong>University</strong> School <strong>of</strong> Medicine, Indianapolis, IN; # Cincinnati Children's Hospital Medical Center,<br />
Cincinnati, OH<br />
Transforming Growth Factor beta (TGFβ) ligands function during the onset and progression <strong>of</strong> aortic valve<br />
calcification remains obscure as both contradictory elevated and suppressed TGFβ signaling has been found in<br />
patients <strong>of</strong> calcific aortic valve disease (CAVD). Here, using in vitro micromass cultures <strong>of</strong> mouse valve<br />
precursor cells (tsA58-AVM) we showed by complimentary realtime QPCR and synthetic luciferase reporter<br />
assays that TGFβ3 can upregulate Sry-related high-mobility-group box 4 (SOX4) transcription. Sox4 is initially<br />
expressed within the endocardially-derived tissue <strong>of</strong> both the outflow tract and atrioventricular canal during<br />
development, but persists within adult valve mesenchyme. Moreover, TGFβ3-dependent regulation <strong>of</strong> Sox4 can<br />
be blocked by a small molecule inhibitor <strong>of</strong> SMAD3 (SIS3), indicating that SMAD3 acts as a downstream<br />
mediator <strong>of</strong> TGFβ3-dependent Sox4 induction. Significantly, in situ hybridization revealed that TGFβ3 is the<br />
most prevalent TGFβ is<strong>of</strong>orm in postnatal heart valves. Histology demonstrated that bothTgfb3 -/- fetal (E14.5)<br />
and Smad3 -/- adult (6 month old) valves were abnormally thickened. There was no calcification in the aortic<br />
valves <strong>of</strong> Smad3 -/- mice. Correlative analysis <strong>of</strong> human valve tissues via immunohistochemistry (5 controls, 8<br />
CAVD, 4 non-calcific aortic valve disease) demonstrated diffused expression <strong>of</strong> SOX4 in the control tissues,<br />
which was extinguished in the diseased tissue specimens. Interestingly, the expression <strong>of</strong> SOX4 around the<br />
calcific nodules remains upregulated within the diseased aortic valves from CAVD patients. Consistently, Sox4<br />
siRNA knockdown in tsA58-AVM cells significantly decreased calcification. Overall, our results indicate that<br />
TGFβ3-dependent and SMAD3-mediated regulation <strong>of</strong> SOX4 may play an important role in valve remodeling<br />
and aortic valve calcification. Since small molecule inhibitor/s <strong>of</strong> SMAD3 could be used as a therapeutic<br />
approach, these data provide novel basic science rationale with potential to translate into different therapies for<br />
patients <strong>of</strong> CAVD.<br />
8.8 Nuclear exclusion <strong>of</strong> Sox9 in valve interstitial cells is associated with calcific phenotypes in heart<br />
valves<br />
Danielle J. Huk 1,2 , Harriet Hammond 2 , Robert. B. Hinton 3 , and Joy Lincoln 2<br />
1 Molecular and Cellular Pharmacology Graduate Program, <strong>University</strong> <strong>of</strong> Miami Miller School <strong>of</strong> Medicine, Miami,<br />
FL, 2 Center <strong>of</strong> Cardiovascular and Pulmonary Research, The Research Institute at Nationwide Children’s<br />
Hospital, Department <strong>of</strong> Pediatrics, The Ohio State <strong>University</strong>, Columbus, OH, 3Division <strong>of</strong> Cardiology, The<br />
Heart Institute, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH,<br />
Calcific aortic valve disease (CAVD) is a major public health problem with no effective treatment available other<br />
than valve replacement surgery. Despite the clinical significance, the mechanisms <strong>of</strong> pathogenesis are poorly<br />
understood. Normal valve structures are composed <strong>of</strong> organized layers <strong>of</strong> extracellular matrix interspersed with<br />
valve interstitial cells (VICs) that share molecular phenotypes with cartilage, including Col2a1. In contrast,<br />
calcified valves are characterized by mineralized matrix and increased expression <strong>of</strong> osteogenic genes including<br />
Spp1. We have shown that Sox9 is required in vivo to promote the cartilaginous matrix and prevent calcification<br />
<strong>of</strong> healthy valves by directly activating Col2a1 and suppressing Spp1 in the nucleus. In this study, we show that<br />
in aortic valves from non-diseased subjects, Sox9 expression is restricted to the nuclei <strong>of</strong> VICs, while nuclear<br />
expression is downregulated and notably cytoplasmic in tissue collected from CAVD patients. Usin g a<br />
pharmacological approach, we show that Sox9-mediated calcification as a result <strong>of</strong> nuclear exclusion can be<br />
induced by retinoic acid (RA) treatment in vivo and in vitro. This decrease in nuclear Sox9 localization is<br />
associated with decreased Col2a1 expression and transactivation, activation <strong>of</strong> the Gene Ontology ‘bone<br />
development’ program and matrix mineralization. Ongoing studies include defining the signaling pathways that<br />
maintain Sox9 nuclear localization to prevent CAVD in healthy valves. Together, these data suggest that Sox9<br />
nucleocytoplasmic shuttling <strong>of</strong> Sox9 plays a role in valve pathogenesis and provides insights to aid in the<br />
development <strong>of</strong> new therapeutic strategies for CAVD.<br />
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8.9 Runx2 is<strong>of</strong>orms have divergent roles in development and pathology in the heart<br />
Andre L.P. Tavares, Jesse A. Brown, Katarina Dvorak and Raymond B. Runyan<br />
Department <strong>of</strong> Cellular and Molecular Medicine, <strong>University</strong> <strong>of</strong> Arizona, Tucson, Arizona, U.S.A. 85724<br />
Formation <strong>of</strong> atrioventricular (AV) valves in the heart begins with an epithelial-mesenchymal cell transition<br />
(EMT). In the exploration <strong>of</strong> EMT, we investigated the presence <strong>of</strong> the transcription factor, Runx2, in the AV<br />
canal. Immunostaining and PCR showed that expression coincided with the onset <strong>of</strong> EMT. siRNA treatment <strong>of</strong><br />
AV canal cultures established that it was required for EMT and that loss specifically prevented the early cell<br />
separation step <strong>of</strong> EMT. Sequence analysis showed that the Runx2 is<strong>of</strong>orm present was Runx2-I and that this<br />
is<strong>of</strong>orm was regulated buy TGFβ2 but not BMP. Inhibition <strong>of</strong> Runx2-I with siRNA produced a loss <strong>of</strong><br />
mesenchymal cell markers. Comparison with siRNA towards Snail2, suggests that the two EMT transcription<br />
factors diverge in their regulation <strong>of</strong> downstream components. To determine whether the activity <strong>of</strong> Runx2-I was<br />
conserved in other EMT events, we examined human tissue samples in the progression <strong>of</strong> esophageal<br />
adenocarcinoma. Runx2-I was e xpressed coincident with the transition from columnar epithelia (Barrett’s<br />
Esophagus) to dysplasia, suggesting a conserved role in early EMT. The Runx2-II is<strong>of</strong>orm is associated with<br />
bone and calcifying tissues. To compare is<strong>of</strong>orm activities, we examined Runx2 is<strong>of</strong>orm expression in whole<br />
valve cultures from chicks and in micromass cultures <strong>of</strong> mouse valvular mesenchymal cells. Runx2-II inceases<br />
in culture consistent with expression <strong>of</strong> calcification markers including osteopontin and matrix gla protein and<br />
with positive staining for calcium. Together, the data show a requirement for TGFβ-regulated Runx2-I in EMT<br />
and BMP-regulated Runx2-II with calcification and calcified tissues. Supported by NHLBI HL82851 and Edwards<br />
Life Sciences.<br />
8.10 3D cardiac valve tissue reconstruction and quantitative evaluations <strong>of</strong> tissue & cell phenotypes in<br />
the Pdlim7 knock-out mouse.<br />
Rudyard W. Sadleir 1 , Robert W. Dettman 1 , Jennifer Krcmery 1 , Brandon Holtrup 1 , and Hans-Georg Simon 1<br />
1<br />
Department <strong>of</strong> Pediatrics, Northwestern <strong>University</strong> Feinberg School <strong>of</strong> Medicine, and Children’s Memorial<br />
Research Center, <strong>Chicago</strong>, IL, USA<br />
The actin binding protein Pdlim7 is part <strong>of</strong> a family <strong>of</strong> proteins involved in regulating actin dynamics. Pdlim7 is<br />
expressed throughout cardiac development and into adulthood, and inactivation in zebrafish and mouse models<br />
results in cardiac valve malformations. A recent model for atrio-ventricular (AV) valve formation and maturation<br />
describes the differential contribution <strong>of</strong> epicardially- and endocardially-derived cells to specific leaflets <strong>of</strong> the<br />
mouse valvuloseptal complex. Using the Pdlim7 knock-out mouse, we investigated whether the aberrant<br />
morphology <strong>of</strong> the adult mitral valve is caused by the distinct lineages that shape the lateral and septal leaflets.<br />
We integrate 3D heart valve reconstructions with novel methods to quantitatively test morphological differences<br />
between wildtypes and mutants. The quantitative analysis <strong>of</strong> valve shape demonstrates that our method can<br />
identify subtle, statistically significant morphological changes following loss <strong>of</strong> Pdlim7. From Pdlim7’s role in<br />
actin dynamics, it is possible that the valve tissue difference is the result <strong>of</strong> aberrant cellular migration and/or<br />
cellular shape. To investigate this, we present a 3D method developed to measure cell migration behavior, and<br />
introduce the field <strong>of</strong> 3D geometric morphometrics to studies <strong>of</strong> morphogenesis and statistically evaluate the<br />
patterns <strong>of</strong> cell shape diversity. This work in mice suggests Pdlim7 functions in AV valve remodeling and<br />
maturation at late embryonic and postnatal stages corresponding with the arrival <strong>of</strong> cells from the epicardial<br />
lineage. These findings are important because they describe a mouse model that links a novel gene (Pdlim7) to<br />
post-natal valve maturation and growth.<br />
8.11 Elastic Fiber Defects In Emilin Deficient Aortic Valves Result In Early Bmp-Dependent<br />
Angiogenesis And Late Tgfβ-Dependent Fibrosis<br />
Charu Munjal (1), Amy M. Opoka (1), Giorgio M. Bressan (2), Robert B. Hinton (1)<br />
(1) Division <strong>of</strong> Cardiology, the Heart Institute, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio; (2)<br />
Department Biomedical Science, <strong>University</strong> <strong>of</strong> Padua, Padua, Italy<br />
Emilin-1 (Elastin Micr<strong>of</strong>ibril Interface-Located protein) is an elastin binding protein that regulates elastogenesis<br />
and inhibits TGF-β signaling. Emilin-1 is expressed in the developing and mature heart valves, and Emilin-1<br />
deficiency results in elastic fiber assembly defects. Aortic valve disease (AVD) is characterized by elastic fiber<br />
fragmentation, fibrosis and aberrant angiogenesis. We hypothesized that dysregulation <strong>of</strong> canonical and noncanonical<br />
TGF-β signaling induces fibrosis and angiogenesis respectively in the Emilin-1 deficient (Emi1−/−)<br />
model <strong>of</strong> AVD. The expression <strong>of</strong> Emilin-1 binding proteins elastin and fibulin-5 (an angiostatic factor) was<br />
decreased at the adult and aged stages. In addition, the Emi1−/− aortic valve displays a marked increase in<br />
161
angiogenic factors (VEGF) at the juvenile stage and fibrosis at the aged stage only. Interestingly, pSmad1/5/8,<br />
p-Erk1/2, and elastase expression were increased at early stages, while p Smad2 and macrophage expression<br />
was increased at the late stage only. Although aged Emi1−/− valve tissue did not show calcification, cultured<br />
Emil1−/− valve interstitial cells do calcify faster and more abundantly than control cells in response to<br />
osteogenic stimuli. Animals die prematurely at the age <strong>of</strong> 14-18 months. Echocardiography showed normal<br />
ventricular function in Emil1−/− adults, and marked ventricular dysfunction in aged mutants. These findings<br />
identify the Emil1−/− mouse as a model <strong>of</strong> latent progressive AVD, similar to human disease, and implicate early<br />
BMP signaling dysregulation in the angiogenic response and late TGF-β signaling dysregulation in the<br />
fibrocalcific and inflammatory responses.<br />
8.12 A Spatial Transcriptional Pr<strong>of</strong>ile <strong>of</strong> the Chick and Mouse Endocardial Cushions Identify Novel<br />
Regulators <strong>of</strong> Endocardial EMT in vitro<br />
Daniel M. DeLaughter 1 , Jamille Y. Robinson 2 , H. Scott Baldwin 1, 3 , J. G. Seidman 4 , Joey V. Barnett 2<br />
1 Department <strong>of</strong> Cell and Developmental Biology, Vanderbilt <strong>University</strong> Medical Center, Nashville, TN, USA.<br />
2 Department <strong>of</strong> Pharmacology, Vanderbilt <strong>University</strong> Medical Center, Nashville, TN, USA. 3 Department <strong>of</strong><br />
Pediatrics, Vanderbilt <strong>University</strong> Medical Center, Nashville, TN, USA. 4 Department <strong>of</strong> Genetics, Harvard<br />
Medical School, Boston, MA<br />
Early in valvulogenesis a subpopulation <strong>of</strong> endocardial cells overlaying the cushions <strong>of</strong> the atrial-ventricular<br />
canal (AVC) and outflow tract (OFT) undergoes an epithelial-to-mesenchymal transformation (EMT). We<br />
developed a strategy to identify genes important in endocardial EMT using a spatial transcriptional pr<strong>of</strong>ile. AVC,<br />
OFT, and ventricles (VEN) were isolated from chick and mouse embryos at comparable stages <strong>of</strong> development<br />
(chick HH18; mouse E11.0) when EMT occurs in the AVC and OFT, but not VEN. RNA sequencing analysis <strong>of</strong><br />
gene expression between these three regions was performed. 347 genes in the chick (n=1) and 225 genes in<br />
the mouse (n=2) were enriched 1.25 fold in the cushions. 18.2% mouse cushion enriched genes (41/225) had<br />
known roles in valve development. Gene ontology (GO) analysis <strong>of</strong> these gene lists revealed biological<br />
processes associated with EMT (cell adhesion, cell movement, ECM organization) that were conserved<br />
between chick and mouse. Gene regulatory networks (GRN) generated from cushion enriched gene lists<br />
identified TGFb and BMP as nodal points. Five candidate genes whose role in endocardial EMT in vitro was<br />
unknown, HAPLN1, ID1, FOXP2, TPBG, and MEIS2, were selected for analysis in an in vitro collagen gel<br />
assay. Knockdown <strong>of</strong> each resulted in an inhibition <strong>of</strong> EMT, functionally validating these genes as regulators <strong>of</strong><br />
EMT in vitro. Our spatial transcriptional pr<strong>of</strong>iling strategy not only yielded gene lists which reflected the known<br />
biology <strong>of</strong> the system, by GO and GRN analysis, but accurately identified and validated novel candidate genes<br />
with previously undescribed roles in endocardial cell EMT in vitro.<br />
8.13 Periostin/Filamin A: a candidate central regulatory mechanism for valve fibrogenesis and matrix<br />
compaction.<br />
Misra, S, Ghatak, S, Markwald, RR, Norris, RA<br />
Regenerative Medicine and Cell Biology and Anatomy, MUSC, Charleston, SC 29425.<br />
We have previously shown that periostin (PN), a matricellular protein secreted by prevalvular cushion<br />
mesenchyme promotes cell autonomous, fibrogenic differentiation and compaction <strong>of</strong> collagenous matrix into<br />
mature valve leaflets and cusps. Loss <strong>of</strong> periostin inhibits differentiation into fibroblast lineages, collagen<br />
secretion and matrix compaction resulting in elongated and myxomatous valves. With respect to mechanism(s),<br />
PN promotes matrix compaction by directly binding to collagen or indirectly by binding to β integrins and altering<br />
cytoskeleton organization that modify matrix compaction. Specifically, to test the latter mechanism, we initiated<br />
signaling studies to determine if PN promoted valve compaction and maturation by activating β3(β1) integrinassociated<br />
signaling and if such activation included phosphorylation <strong>of</strong> filamin-A (FLNA), an actin-binding<br />
protein expressed in valve interstitial fibroblasts. IP data, western blots and immunostaining indic ated that PN<br />
binding to β 3 integrin specifically promoted phosphorylation <strong>of</strong> FLNA (2152) by activating cdc42 and pak1<br />
kinases and that 2152 phosphorylation site <strong>of</strong> FLNA occurred near two point mutations (P673Q,G228R) found in<br />
patients with degenerative (myxomatous) valve diseases. Silencing periostin inhibited FLNA (2152)<br />
phosphorylation which correlated with reduced potential <strong>of</strong> prevalvular interstitial cells to compact collagen gels.<br />
Similarly, we also found that introducing the P673Q and G228R mutations into these cells also inhibited<br />
collagen compaction and the phosphorylation <strong>of</strong> FLNA (2152). Supported by NHLBI, AHA, NCRR and Leducq<br />
Mitral Transatlantic Network<br />
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8.14 Fibulin-1 regulation <strong>of</strong> cell cycle progression in developing cardiac valves<br />
Keerthi Harikrishnan 1 , Marion A. Cooley 1 , Waleed O. Twal 1 , Victor M. Fresco 1 , Christine B. Kern 1 , Eugenia<br />
Broude 2 and W. Scott Argraves 1<br />
1 Department <strong>of</strong> Regenerative Medicine and Cell Biology, Medical <strong>University</strong> <strong>of</strong> South Carolina, Charleston, SC<br />
29425; 2 Department <strong>of</strong> Pharmaceutical and Biomedical Sciences, South Carolina College <strong>of</strong> Pharmacy<br />
<strong>University</strong> <strong>of</strong> South Carolina, Columbia, SC 29208<br />
Fibulin-1 (Fbln1) is a component <strong>of</strong> the extracellular matrix <strong>of</strong> endocardial cushions and adult cardiac valves, but<br />
its function in the process <strong>of</strong> cardiac valvulogenesis is not known. Analysis <strong>of</strong> Fbln1 null embryonic hearts<br />
revealed that aortic and pulmonary valves are smaller (30%; p
studied before and after (3-6 months) procedures which increased AV flow. Results: In unbalanced AV canal<br />
defects, the ventricles went from very hypoplastic 9.8 – 19.4 cc/m 2 (-7.1 to -4.2 SEM) to normal range 32 – 45<br />
cc/m 2 (-1.2 to -0.1 SEM). In pulmonary atresia with intact ventricular septum, right ventricular volumes 2.5 ± 1.1<br />
cc/m 2 (-5.1 ± 2.5 SEM) grew to 19.4 ± 4.6 cc/m 2 (-1.5 ± 1.9 SEM). Other possible mechanisms (high wall stress<br />
or significant retrograde flow from semilunar valve regurgitation) did not produce catch-up ventricular growth.<br />
Conclusions: 1) Congenital heart defects can serve as models for basic developmental questions. 2)<br />
Increased AV valve flow provides the ventricular growth signal. 3) Operations which increased AV valve flow<br />
induced catch-up growth <strong>of</strong> hypoplastic ventricles.<br />
8.17 MAPK and twist1 mediate scleraxis expression to regulate chondroitin sulfate proteoglycans in<br />
developing heart valves.<br />
Damien Barnette 1 and Joy Lincoln 2 .<br />
1 Molecular and Cellular Pharmacology Graduate Program, <strong>University</strong> <strong>of</strong> Miami Miller School <strong>of</strong> Medicine, Miami,<br />
FL USA. 2 Center <strong>of</strong> Cardiovascular and Pulmonary Research, Department <strong>of</strong> Pediatrics, The Ohio State<br />
<strong>University</strong>, Columbus, OH, USA.<br />
In contrast to normal valves, diseased myxomatous mitral valves (MMV) are pathologically thickened and<br />
display defects in connective tissue organization, associated with excess chondroitin sulfate proteoglycan<br />
(CSPG) deposition and functional prolapse. Despite the clinical significance, the signaling pathways required for<br />
regulation <strong>of</strong> CSPG deposition in developing and mature heart valves are not fully understood. The basic helixloop-helix<br />
(bHLH) transcription factor scleraxis (scx) is highly expressed in developing valve structures and null<br />
mice display disorganized connective tissue and attenuated CSPG expression. In support, scx gain-<strong>of</strong>-function<br />
in vitro increases CSPG expression, and additional transfection studies <strong>of</strong> scx mutant constructs in C3H10T1/2<br />
cells suggest that the DNA-binding domain is required to promote CSPG expression. To determine the<br />
molecular mechanisms <strong>of</strong> scx-mediated CSPG regulation, we employed an established in vitro chicken valve<br />
precursor (VP) cell culture system. As studies have shown that MAPK signaling can influence scx, we treated<br />
VP cells with adenovirus targeting MEK1/2. Forty-eight-hour treatment with constitutively active MEK1/2<br />
adenovirus (AdV-caMEK1/2) significantly decreased scx (4.8-fold) and CSPG expression, while dominant<br />
negative MEK1/2 adenovirus (AdV-dnMEK1/2) resulted in an increase in scx (9.5-fold) and CSPG expression.<br />
As ERK1/2 phosphorylation sites are absent on scx, we hypothesized that MAPK regulation is indirect; and<br />
pervious studies suggest bHLH repressor twist1 as a signaling mediator. In our system, AdV-caMEK1/2<br />
treatment dramatically increased twist1 protein levels, and 48-hr AdV-twist1 treatment led to a significant<br />
decrease (4-fold) in scx expression. Ongoing studies will examine the role <strong>of</strong> MAPK-twist1-scx signaling<br />
pathways in MMV disease in human patients and mouse models.<br />
8.18 Filamin-A Functions as a Rheostat to Control Synthesis <strong>of</strong> Matrix Proteins During Valve<br />
Development and Disease<br />
Annemarieke de Vlaming 1 , Kimberly Sauls 1 , Katherine Williams 1 , Andy Wessels 1 , Robert A. Levine 2 , Susan A<br />
Slaugenhaupt 3 , Roger R. Markwald 1 , Russell A. Norris 1*<br />
1 Department <strong>of</strong> Regenerative Medicine and Cell Biology, School <strong>of</strong> Medicine, Cardiovascular Developmental<br />
Biology Center, Children’s Research Institute, Medical <strong>University</strong> <strong>of</strong> South Carolina, Charleston, SC, 29425,<br />
USA. 2 Noninvasive Cardiac Laboratory, Department <strong>of</strong> Medicine, Massachusetts General Hospital, Boston,<br />
Massachusetts, 02114, USA. 3 Center for Human Genetic Research, Massachusetts General Hospital/Harvard<br />
Medical School, Boston, Massachusetts, 02114, USA<br />
Linkage and sequencing studies <strong>of</strong> patients with myxomatous valvular dystrophy revealed causal mutations in<br />
the Filamin-A gene. These studies defined Filamin-A as integral to valve structure and function. However, the<br />
mechanisms by which Filamin-A regulates valve cell biology are unknown. Herein we demonstrate that Filamin-<br />
A can function as a rheostat to precisely control the amount and accumulation <strong>of</strong> extracellular matrix proteins<br />
during valve development. Our studies define that Filamin-A, through its interactions with the MAPK signal<br />
transducer, r-Ras can regulate the phospho-status <strong>of</strong> pErk1/2. We further define that a normal function <strong>of</strong><br />
pErk1/2 is to suppress nuclear translocation <strong>of</strong> pSmad3 by phosphorylating the Smad3 linker region. Filamin-A<br />
deficient valves show a near complete loss <strong>of</strong> r-Ras and pErk1/2 activity and a subsequent increase in Smad3<br />
C-terminal phosphorylation and nuclear accumulation. By neonatal timepoints, a significant increase in nuclear<br />
pSmad3 levels is observed concomitant with increased production <strong>of</strong> fibrillar extracellular matrix proteins.<br />
Dysregulated matrix synthesis in the Filamin-A deficient mouse continues throughout postnatal life progressing<br />
to a myxomatous valvular dystrophy by 2 months <strong>of</strong> age. Thus, our studies indicate matrix synthesis during<br />
valve development may be tightly regulated through a novel yin-yang interaction between MAPK pathways and<br />
164
TGFb/Smad3 pathways and are dependent on Filamin-A acting as a rheostat. As Filamin-A point mutations<br />
causal to human myxomatous valvular dystrophy occur in the r-Ras binding domain, our studies suggest new<br />
mechanisms by which matrix is synthesized during valve development as well as providing insight into<br />
mechanisms underlying degenerative cardiac valve diseases.<br />
8.19 Differentiation <strong>of</strong> heart valve progenitor cells is dependent on communication between periostinintegrin<br />
and hyaluronan-induced signaling<br />
Ghatak, S 1 , Misra, S 1 , Norris, RA 1 , Moreno-Rodriguez, RA 1 , Sugi,Y 1 , Hascall, VC 2 , H<strong>of</strong>fman, S 1 , Markwald, RR 1<br />
1 Regenerative Medicine and Cell Biology and Anatomy, MUSC, Charleston, SC 29425. 2 Cleveland Clinic, Ohio.<br />
Periostin (PN) is a fasciclin-related protein secreted by prevalvular mesenchyme <strong>of</strong> the ventricular inlets and<br />
outlets and interstitial cells <strong>of</strong> the ventricular myocardium. We have previously shown that PN promotes cell<br />
autonomous, fibrogenic-differentiation and compaction <strong>of</strong> collagenous matrix into mature valve. Loss <strong>of</strong> periostin<br />
inhibits differentiation into fibroblast lineages, collagen secretion and matrix compaction resulting in enlongated<br />
valves with potential for regurgitation. we investigated PN, as a matricellular protein, has potential to signal for<br />
valve compaction and maturation by activating β3/(β1) integrin-associated pathways and determined if such<br />
activation has cross-talk with hyaluronan signaling. Our recent studies demonstrate that: (1) PN increases<br />
hyaluronan secretion via PN/integrin/PI3K signaling in embryonic valve cells, (2) PN-induced signaling<br />
communicates with hyaluronan/CD44 interacted signaling, (3) PN interacts with specific integr in β3 downstream<br />
PI3K/AKT mediated cell survival to stimulate adhesion/growth and AV cell survival, (3) PN/integrin-β1 interaction<br />
activate cytoskeletal associated filamin-A, and (5) periostin and hyaluronan are co-expressed in wild type E16.5<br />
mouse valve sections, whereas hyaluronan expression is suppressed in E16.5 periostin null mouse valve<br />
sections. Together, these findings indicate that PN and hyaluronan can regulate cytoskeletal changes required<br />
for valve maturation by triggering signaling mechanisms that activate cytoskeletal FLNA. Supported by NHLBI,<br />
AHA, NCRR and Leducq Mitral Transatlantic Network.<br />
8.20 Bicuspid Aortic Valve Leaflets Experience Increased Stretch which Activates Inflammatory Gene<br />
Expression.<br />
Kai Szeto 1,2 , Katrina Carrion 1 , Thomas Gallegos 3 , Andrew Hollands 4 , Jeffrey Dyo 1 , Vishal Patel 1 , Sanjay Nigam 3 ,<br />
Peter Pastuszko 5 , Victor Nizet 4 , Juan Lasheras 2 , Vishal Nigam 1<br />
1 Department <strong>of</strong> Pediatrics (Cardiology), <strong>University</strong> <strong>of</strong> California San Diego; 2 Department <strong>of</strong> Mechanical<br />
Engineering, <strong>University</strong> <strong>of</strong> California San Diego; 3 Department <strong>of</strong> Pediatrics, <strong>University</strong> <strong>of</strong> California San Diego;<br />
4 Department <strong>of</strong> Pediatrics and School <strong>of</strong> Pharmacy, <strong>University</strong> <strong>of</strong> California San Diego; 5 Department <strong>of</strong> Surgery<br />
(Cardiothoracic Surgery), <strong>University</strong> <strong>of</strong> California San Diego<br />
Aortic valve calcification is the third leading cause <strong>of</strong> adult heart disease and the most common form <strong>of</strong> acquired<br />
valvular disease in developed countries. The risk factor most closely linked to calcific aortic stenosis is bicuspid<br />
aortic valve (BAV). Examination <strong>of</strong> calcified aortic valve leaflets demonstrates that inflammatory changes are<br />
involved. There is no effective treatment other than valve replacement. Additional understanding <strong>of</strong> the<br />
molecular pathogenesis <strong>of</strong> aortic valve calcification is required to develop effective medical treatments for this<br />
disease. To determine if the fused leaflet <strong>of</strong> BAVs experience more stretch than tricuspid aortic valves (TAVs),<br />
we performed ex vivo experiments comparing porcine TAV valves to surgically created porcine BAVs. We found<br />
that BAVs experience 25% stretch in the radial direction as compared to 5% for TAVs. To determine if increased<br />
stretch altered gene expression in human aortic valve interstitial cells (AVICs), we used microarrays to pr<strong>of</strong>ile<br />
the RNA expression pr<strong>of</strong>iles between AVICs exposed to static condition or 14% stretch at 1hz for 24h.<br />
INTERLEUKIN1B (IL1B) and MATRIX METALLOPROTEINASE-1 (MMP1) were among the most stretch<br />
unregulated genes. These genes are found at higher levels in calcified aortic valves. Additional INTERLEUKIN<br />
and MATRIX METALLOPROTEINASE family members were also unregulated by stretch. The expression<br />
changes were confirmed by qPCR. Our data demonstrates that the fused leaflet <strong>of</strong> BAV experiences increased<br />
stretch and that this stretch is sufficient to activate some <strong>of</strong> the inflammatory genes that are expressed at higher<br />
levels in calcified aortic valves.<br />
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8.21 4-D Shear Stress Maps <strong>of</strong> the Developing Heart using Doppler Optical Coherence Tomography<br />
Lindsy M. Peterson 1 , Michael W. Jenkins 1 , Shi Gu 1 , Lee Barwick 1 , Yong-Qiu Doughman 2 , Michiko Watanabe 2 ,<br />
and Andrew M. Rollins 1<br />
1 Department <strong>of</strong> Biomedical Engineering, Case Western Reserve <strong>University</strong>, Cleveland, Ohio 44106<br />
2 Department <strong>of</strong> Pediatrics, Case Western Reserve <strong>University</strong>, Cleveland, Ohio 44106<br />
Accurate imaging and measurement <strong>of</strong> hemodynamic forces is vital for investigating how physical forces acting<br />
on the embryonic heart are transduced and influence developmental pathways. However, making these<br />
measurements in the functioning embryonic heart is difficult due to its diminutive size and rapidly changing,<br />
pulsatile blood flow. Of particular importance is blood flow-induced shear stress, which influences gene<br />
expression by endothelial cells and potentially leads to congenital heart defects through abnormal heart looping,<br />
septation, and valvulogenesis. Previous efforts to measure the shear stress on the endocardium have not<br />
succeeded in mapping these patterns in 3-D and 4-D. Using 4-D structural and Doppler OCT, we are able to<br />
accurately measure the blood flow in the heart tube in vivo and to map endocardial shear stress throughout the<br />
heart cycle under physiological conditions. These shear stress measurements were compared with<br />
immunostaining patterns <strong>of</strong> KLF2, a shear responsive protein. KLF2 was highly expressed along the inner<br />
curvature <strong>of</strong> the outflow tract which corresponds well with our shear stress measurements. These combined<br />
measurements <strong>of</strong> the beating heart will be valuable in determining the influence <strong>of</strong> shear force on normal and<br />
abnormal development and may be sensitive enough to detect the earliest signs <strong>of</strong> congenital heart defects. For<br />
the first time, we can directly measure the pattern <strong>of</strong> shear stress on the endocardium <strong>of</strong> the developing heart<br />
tube in 4-D, which will enable precise titration <strong>of</strong> experimental perturbations and accurate correlation <strong>of</strong> shear<br />
with expression <strong>of</strong> molecules critical to heart development. (RO1HL083048, R01HL095717, T32EB007509,<br />
C06RR12463-01)<br />
8.22 The origin and the role <strong>of</strong> hematopoietic-derived cells in embryonic heart valve development<br />
Zoltan Hajdu, Agnes Nagy-Mehesz, Russel A. Norris, Roger R. Markwald, Richard P. Visconti<br />
Dept. Regenerative Medicine and Cell Biology, Medical <strong>University</strong> <strong>of</strong> South Carolina, Charleston, SC, USA<br />
Our group has previously described the presence <strong>of</strong> CD45 + cells in the developing cardiac valves and their<br />
contribution to homeostasis and pathological remodeling <strong>of</strong> the post-natal valves. We have shown that CD45 +<br />
cells in the post-natal valves originate from the hematopoietic compartment <strong>of</strong> the bone marrow. However, the<br />
source <strong>of</strong> these cells in the embryonic valves is unclear. To determine whether CD45 might be expressed by<br />
endocardially-derived cells that populate the endocardial cushions, or is expressed only on valve cells <strong>of</strong> extracardiac<br />
origin, chick cushions from stages prior (ED3) and after (ED5) CD45 + cells populate them in vivo were<br />
explanted onto collagen gels and cultured for up to three days. Using this approach, we never detected CD45 +<br />
cells in the ED3 explants, suggesting that CD45 is not expressed on endocardially-derived cushion cells. This<br />
finding is supported by our quail-chick chimera studies, where the quail aorta was transplanted into chicken<br />
embryos. QH1 + cells were detected interspersed between host CD45 + cells in the cushion mesenchyme,<br />
highlighting the ability <strong>of</strong> embryonic hematopoietic cells to migrate into the developing heart valves. Next, to<br />
begin to evaluate whether CD45 + cells might contribute to valve development, we used CCN1-null mice in order<br />
to prevent the invasion <strong>of</strong> CD45 + cells into the developing cushions. Along with the already described<br />
malformations in CCN1-null hearts, we observed significantly reduced CD45 + cell numbers in the cushions.<br />
Collectively, our findings suggest that hematopoietic-derived (CD45 + ) cells migrate into the developing valves<br />
and perturbation <strong>of</strong> their invasion may contribute to the cardiac malformations.<br />
8.23 The role <strong>of</strong> BMP in differentiation and lineage restriction <strong>of</strong> AV endocardial cushion cells<br />
Yukiko Sugi, 1 Kei Inai, 1* Bin Zhou, 2 Mark Mercola, 3 Roger Markwald, 1 YiPing Chen, 4 Yuji Mishina, 5 Jessica<br />
Burnside 1<br />
1 Department <strong>of</strong> Regenerative Medicine and Cell Biology, Medical <strong>University</strong> <strong>of</strong> South Carolina, 2 Albert Einstein<br />
College <strong>of</strong> Medicine <strong>of</strong> Yeshiva <strong>University</strong>, 3 Sanford-Burnham Medical Research Institute, 4 Tulane <strong>University</strong>,<br />
5 <strong>University</strong> <strong>of</strong> Michigan, *present address: Tokyo Women’s Medical <strong>University</strong><br />
Formation and distal outgrowth <strong>of</strong> the endocardial cushion mesenchyme in the atrioventricular (AV) canal is the<br />
first and essential morphogenetic event in cardiac valvuloseptal morphogenesis. AV endocardial cushion<br />
mesenchymal cells (ECMCs) likely have the potential to abnormally differentiate into other mesodermal cell<br />
lineages. BMPs are multifaceted regulators that regulate cell differentiation in a cell-type dependent fashion. Our<br />
current research tested the hypothesis that BMP-2 signaling accomplishes two-fold functions <strong>of</strong> i) promoting<br />
ECMC differentiation into pre-valvular fibroblasts and ii) inhibiting differentiation <strong>of</strong> alternative mesodermal cell<br />
166
fates via intersection with other cell fate determinants like Notch pathway. This hypothesis is supported by our<br />
data that BMP-2 ligand and BMP type I receptors, Alk3 and Alk2, as well as notch receptors Notch ½ are<br />
expressed in ECMCs during AV cushion maturation and that BMP-2 induces expression <strong>of</strong> Notch ½ and N otch<br />
pathway intermediates in the ECMC aggregate culture. BMP-2 induces the valvulogenic phenotype including<br />
extracellular matrix (ECM) protein expression and cellular migration and also inhibits expression <strong>of</strong> alternative<br />
mesodermal lineage markers in chick ECMC cultures. To complement this in vitro finding, we also tested the<br />
effect <strong>of</strong> gain and loss <strong>of</strong> BMP signaling in AV cushion mesenchyme in vivo using an endocardium-specific credriver<br />
line. Alk3 endocardial conditional knock-out mice die by embryonic day 12.5 and have defects in<br />
endocardial cushion development and ECM protein expression in ECMCs, whereas endocardial/endocardial<br />
cushion specific constitutive up-regulation <strong>of</strong> Alk3 results in enlarged endocardial cushions with up-regulated<br />
pSmad 1/5/8 expression and abundant ECM protein expression in ECMCs.<br />
8.24 Glycerophosphocholines and Sphingolipids are Compartmentalized within Ovine and Human<br />
Aortic Valve Substructures and are Differentially Regulated during Ovine Transitional Circulation<br />
Peggi M. Angel, 1 Ahmed Bayoumi, 2 John E. Mayer, 2 Brett A. Mettler, 1 David P. Bichell, 1 Yan Ru Su, 1 Richard<br />
M. Caprioli, 1 H. Scott Baldwin 1<br />
1<br />
Vanderbilt <strong>University</strong> Medical Center, Nashville, TN, USA<br />
2<br />
Harvard Medical School, Boston, MA, USA<br />
Introduction: Congenital and acquired aortic valve stenosis (AVS) affects over 10 million people with cellular<br />
mechanisms mostly unknown. Degeneration <strong>of</strong> the valve structure involves atherosclerotic-like processes with<br />
lipid deregulation and calcification triggered by activated valvular interstitial cells (VIC). Since disease-activated<br />
VIC are proposed to recapitulate embryonic programs, we tested the hypothesis that lipids are regulated within<br />
the developing aortic valve (AV). Methods: Ovine AV at prenatal day 5 and adult (each n=3), de-identified<br />
human AV leaflets from pediatric patients with severe AVS (n=2) were examined for lipid distribution using<br />
matrix-assisted laser desorption ionization imaging mass spectrometry. Lipid alteration in AV substructure was<br />
modeled by receiver operating characteristic curve ≥0.8 with spectral sets >100 per each valve region. Valve<br />
extracellular matrix (ECM) was evaluated by Movat’s pentachrome and Verhoeff-Van Geison stain. Coexpression<br />
<strong>of</strong> endothelial differentiation gene 1 (EDG1) and α smooth muscle actin, a marker <strong>of</strong> aVIC, was<br />
investigated by immun<strong>of</strong>luorescence. Results/Conclusions: Glycerophosphocholines and sphingolipids were<br />
spatially restricted and regulated within human and ovine AV substructures. Expression patterns followed ECM<br />
stratification. Lipid signatures from ovine fetal and adult substructures showed alteration in 18 lipid species.<br />
Principle component analysis showed distinct clustering between ovine timepoints. EDG1, the receptor for<br />
sphingosine-1-phosphate, was expressed by VIC at ovine fetal and adult timepoints with decreased expression<br />
in the aortic wall. Lipids followed aberrant ECM in human pediatric AVS. This study defines a novel role for lipids<br />
in valve development and provides a new approach to investigating lipid involvement in AV disease.<br />
8.25 Fibulin-1 regulation <strong>of</strong> cell cycle progression in developing cardiac valves<br />
Keerthi Harikrishnan 1 , Marion A. Cooley 1 , Waleed O. Twal 1 , Victor M. Fresco 1 , Christine B. Kern 1 , Eugenia<br />
Broude 2 and W. Scott Argraves 1<br />
1 Department <strong>of</strong> Regenerative Medicine and Cell Biology, Medical <strong>University</strong> <strong>of</strong> South Carolina, Charleston, SC<br />
29425; 2 Department <strong>of</strong> Pharmaceutical and Biomedical Sciences, South Carolina College <strong>of</strong> Pharmacy<br />
<strong>University</strong> <strong>of</strong> South Carolina, Columbia, SC 29208.<br />
Fibulin-1 (Fbln1) is a component <strong>of</strong> the extracellular matrix <strong>of</strong> endocardial cushions and adult cardiac valves, but<br />
its function in the process <strong>of</strong> cardiac valvulogenesis is not known. Analysis <strong>of</strong> Fbln1 null embryonic hearts<br />
revealed that aortic and pulmonary valves are smaller (30%; p
progression in developing valves and perhaps other tissues where it functions to prevent mitotic catastrophe<br />
leading to cellular senescence. This work was supported from NIH grant HL095067.<br />
8.26 Direct Role <strong>of</strong> Hyperphosphatemia in Vascular and Heart Valve Calcification<br />
Margaret Benny Klimek 1 , Christian Faul 2 and Joy Lincoln 3<br />
1 Molecular, Cell and Developmental Biology Graduate Program, Leonard M. Miller School <strong>of</strong> Medicine, <strong>University</strong> <strong>of</strong> Miami,<br />
Miami, FL. 2 Division <strong>of</strong> Nephrology and Hypertension, Department <strong>of</strong> Medicine, Leonard M. Miller School <strong>of</strong> Medicine,<br />
<strong>University</strong> <strong>of</strong> Miami, Miami, FL. 3 Center <strong>of</strong> Cardiovascular and Pulmonary Research, The Research Institute at Nationwide<br />
Children’s Hospital, Department <strong>of</strong> Pediatrics,The Ohio State <strong>University</strong>, Columbus, Ohio<br />
The incidence <strong>of</strong> vascular and aortic valve calcification is significantly increased in patients with chronic kidney<br />
disease leading to mortality related to cardiovascular events. This pathological calcification has been<br />
associated with hyperphosphatemia and elevated serum levels <strong>of</strong> FGF23, however the direct effects <strong>of</strong> these<br />
circulating factors on valve calcification has not been examined. To address this, pig aortic valve interstitial cells<br />
(pAVICS) and murine aortic valve (mAoV) explants were treated with 1-3mM phosphate-supplemented media or<br />
25-100ng/mL FGF23 for 3-8 days. Following treatment with phosphate, but not FGF23, pAVICs and mAoV<br />
explants cells demonstrate a significant increase in Von Kossa reactivity (213 and 109 fold increase, p
This valve specific miR-21 expression was downregulated when the heart beat was blocked by BDM (myosin<br />
specific ATPase inhibitor), while it was promptly restored by re-initiating the heart beat. These results suggest a<br />
hemodynamic-dependent expression <strong>of</strong> miR-21. Knock down <strong>of</strong> miR-21 by antisense morpholino oligo induced<br />
hypoplastic AVV. Endocardial cells in the valve forming region failed to proliferate in miR-21 morphants,<br />
indicating that miR-21 plays a cell growth-related role in response to the heart beat.<br />
All in all, miR-21 expression triggered by mechanical stresses contributes to zebrafish AVV formation. We also<br />
show updated data on the function <strong>of</strong> miR-21 promoter/enhancer in responding to mechanical stresses.<br />
8.29 W-Loop Of Alpha-Cardiac Actin Is Critical For Heart Function And Endocardial Cushion<br />
Morphogenesis In Zebrafish<br />
Nikki Glenn 1 , Kuo-Kuang Wen 2 , Vikram Kohli 1 , Melissa Mckane 2 , Peter A. Rubenstein 2 , Thomas Bartman 1,3 ,<br />
Saulius Sumanas 1<br />
1 2 3<br />
Cincinnati Children’s Hospital, Cincinnati, OH <strong>University</strong> Of Iowa, Iowa City, IA Nationwide Children’s Hospital<br />
Columbus, OH<br />
Mutations in cardiac actin (ACTC) have been associated with different types <strong>of</strong> cardiac abnormalities in humans<br />
including dilated cardiomyopathy and septal defects. However, it is still poorly understood how altered ACTC<br />
structure affects cardiovascular physiology and results in the development <strong>of</strong> distinct congenital disorders. A<br />
cardiovascular-specific zebrafish mutant (s434) was identified that displays blood regurgitation in a dilated heart<br />
between the atrium and ventricle and lacks endocardial cushion formation. We identified the mutation as a<br />
single nucleotide change in Alpha-Cardiac Actin (actc1a), resulting in a Y169S amino acid substitution. This<br />
mutation affects the W-loop <strong>of</strong> actin, which has been implicated in nucleotide sensing. Our results demonstrate<br />
that s434 mutants show loss <strong>of</strong> polymerized cardiac actin. An analogous mutation in yeast results in rapid<br />
depolymerization <strong>of</strong> F-actin into fragments that are not able to reanneal. This polymerization defect can be<br />
partially rescued both in yeast and zebrafish embryos by phalloidin treatment which stabilizes F-actin.<br />
Physiological analyses reveal that actc1a mutant embryos show defects in cardiac contractility and altered blood<br />
flow within the heart tube. As a result, molecular markers for endocardial cushion formation including notch1b,<br />
nfatc1, versican and klf2a are downregulated or mislocalized in s434 mutants, leading to the absence <strong>of</strong> EC<br />
development. Our study underscores the importance <strong>of</strong> the W-loop for actin functionality and will help us to<br />
understand structural and physiological consequences <strong>of</strong> certain ACTC mutations in human congenital<br />
disorders.<br />
8.30 Olfactomedin-1 activity identifies a cell invasion checkpoint during epithelial-mesenchymal<br />
transition in the embryonic heart.<br />
Alejandro Lencinas 1,2 , Danny C. Chhun 1 , Kelvin P. Dan 1,3 , Kristen D. Ross 1 , and Raymond B. Runyan 1,2 .<br />
Departments <strong>of</strong> Cellular and Molecular Medicine 1 , Pharmacology and Toxicology 2 and Program in Physiological<br />
Sciences 3 . The <strong>University</strong> <strong>of</strong> Arizona, Tucson Arizona 85724.<br />
Endothelia in the atrioventricular (AV) canal <strong>of</strong> the developing heart undergo an epithelial mesenchymal<br />
transition (EMT) and invade the underlying extracellular matrix to begin heart valve formation. Using an in vitro<br />
invasion assay, an extracellular matrix protein found in the heart, Olfactomedin-1 (OLFM1), increases<br />
mesenchymal cell numbers and anti-OLFM1 antibody inhibits normal mesenchymal cell formation. OLFM1<br />
does not alter cell proliferation, migration or apoptosis. Dispersion, but lack <strong>of</strong> invasion in the presence <strong>of</strong><br />
inhibiting antibody, identifies a role for OLFM1 in cell invasion during EMT. To explore OLFM1 activity during<br />
EMT, representative EMT markers were examined. Effects <strong>of</strong> OLFM1 and anti-OLFM1 on cell-cell adhesion<br />
molecules and the transcription factors, Snail-1, Snail-2, Twist1, and Sox-9 are inconsistent with initiation <strong>of</strong><br />
EMT. Transcription factors, Zeb1 and Zeb2, secreted proteases and several mesenchymal cell markers are<br />
regulated by OLFM1 and anti-OLFM1 consistent with regulation <strong>of</strong> invasion. We conclude that OLFM1 is<br />
present and necessary for EMT in the developing heart. Its role in cell invasion and mesenchymal cell gene<br />
expression argues for an invasion checkpoint in EMT where additional signals are required to initiate cell<br />
invasion into the three-dimensional matrix. Incubation <strong>of</strong> several cancer cell lines with exogenous OLFM1<br />
suggests a conserved role in other EMTs.<br />
169
8.31 Twist1 Homodimer and Heterodimer Function in Heart Valve Development<br />
Mary P. Lee 1 , Santanu Chakraborty 1 , Doug B. Spicer 2 , and Katherine E. Yutzey 1<br />
1 The Heart Institute, Cincinnati Children’s Research Foundation, Cincinnati, Ohio<br />
2 Center for Molecular Medicine, Maine Medical Center Research Institute, Scarborough ME<br />
Heart valves develop from extracellular matrix-rich endocardial cushions (ECCs) populated by proliferative,<br />
migratory, and undifferentiated mesenchymal cells. The basic helix-loop-helix transcription factor, Twist1 is<br />
highly expressed in ECCs, but not remodeling valves, and is re-expressed during pediatric and adult heart valve<br />
disease. Twist1 binds to E-box consensus sequences (CANNTG) as a homodimer or a heterodimer with other<br />
bHLH proteins, including E12/E47. Twist1 dimers have differential functions and distinct gene targets in murine<br />
cranial suture and limb development and also in Drosophila mesoderm cell lineage determination. We<br />
hypothesize that Twist1 homo- and heterodimers differentially regulate gene expression and developmental<br />
functions during heart valve development. In previous studies, we identified Tbx20, Cdh11, Col2a1, Sema3C,<br />
Rab39b, and Gadd45a as direct transcriptional targets <strong>of</strong> Twist1 during heart valve development. Differential<br />
binding <strong>of</strong> endogenous Twist1 and E12/E47 to known Twist1 target gene enhancers was observed by chromatin<br />
immunoprecipitation (ChIP) assays. Differential functions <strong>of</strong> Twist1 homo- and heterodimers were examined<br />
using tethered Twist1-Twist1 (TT) and Twist1-E12 (TE) dimer overexpression in cultured cells and transgenic<br />
mice. In MC3T3-E1 preosteoblast cells, expression <strong>of</strong> Twist1 target genes is differentially affected by<br />
transfection <strong>of</strong> TT or TE tethered dimers. In preliminary studies, mice with conditional loss <strong>of</strong> Twist1 or<br />
expression <strong>of</strong> Twist1 tethered homo- and heterodimers have abnormal ECCs and valve morphogenetic defects.<br />
Together these studies provide initial evidence for differential functions <strong>of</strong> Twist1 homo- and heterodimers in<br />
target gene expression and heart valve development.<br />
8.32 The roles <strong>of</strong> Smad8 in calcific aortic valves<br />
Yuka Morikawa 1, 2 , Mahdis Rahmani 1, 2 1, 2<br />
, James F Martin<br />
1 2<br />
Texas Heart Institute, Dept Molecular Physiology, Baylor College <strong>of</strong> Medicine, Houston, TX<br />
Among heart diseases, those affecting the cardiac valves are the most common. Within this group, aortic valve<br />
calcification is especially prevalent as the population ages. The incident <strong>of</strong> aortic valve calcification increases<br />
with age and affects up to 20% <strong>of</strong> the population over the age <strong>of</strong> 80. Although such a prevalent medical<br />
problem, the genetic bases and molecular mechanism underlying how valve diseases develop, in particular how<br />
valve calcification develops, is poorly understood. Among the molecules involved in cardiac valve development<br />
and pathogenesis, Bone Morphogenetic Protein (BMP) signaling has been shown to be essential in aortic valve<br />
development and their misregulation also leads to calcification <strong>of</strong> aortic valves. We have found that systemic<br />
knockout <strong>of</strong> Smad8, one <strong>of</strong> the intercellular components <strong>of</strong> BMP signal transduction pathway, develops valve<br />
defects including disorganization <strong>of</strong> the extracellular matrix layers <strong>of</strong> valves and their calcification in adult<br />
mouse. Smad8 null mice do not develop other major structural cardiac defects making them an ideal model to<br />
study the roles <strong>of</strong> BMP signaling in cardiac valve disease. We have found that in Smad8 null mice, other<br />
members <strong>of</strong> the Smad family, Smad1, 2, and 5, are phosphorylated leading to nuclear localization. Our data<br />
suggests that a negative feedback loop within the Smad pathways regulates development <strong>of</strong> cardiac valves and<br />
their misregulation leads to diseases <strong>of</strong> valves in adults.<br />
170
Author Index<br />
AbdulSater, Zahy 1.24<br />
Abramczuk, Monika S6.6<br />
Abu-Issa, Radwan 6.21<br />
Ackerman, Christine S1.5<br />
Adamoc, Richard S8.5<br />
Adams, Meghan S7.2<br />
Aghajanian, Haig 4.22<br />
Ahrens, Molly 3.26<br />
Akiona, Jennifer 6.8<br />
Alders, M 1.23<br />
Aleksandrova, Anastasiia 6.17<br />
Alexi-Meskishvili, Vladimir S1.4<br />
Allison, Patrick 5.28<br />
Alonzo-Johnsen, Martha 4.15<br />
Amack, Jeffrey 6.13<br />
Amin, Nirav S4.5<br />
Ammirabile, Grazia 5.18<br />
Andelfinger, A 6.26<br />
Anderson, P. 3.22<br />
Anderton, Shane 1.12, 1.15, 1.21<br />
Andrews, William S4.3<br />
Aneas, Ivy S3.5<br />
Ang, SiangYun 7.9<br />
Angel, Peggi 8.24<br />
Aoki, Hiroki 4.27<br />
Apte, Suneel S1.1<br />
Araki, Toshiyuki 1.8<br />
Aránega, Amelia 5.13<br />
Argraves, Scott 3.7, 8.14, 8.25<br />
Arima, Yuichiro S4.4, 4.31, 6.22<br />
Arimura, Takuro 1.26<br />
Arnolds, David S5.4<br />
Arshi, Armin 4.29<br />
Asai, Rieko S4.4, 4.31, 6.22<br />
Astr<strong>of</strong>, Sophie S4.2, 4.17, 7.14<br />
Azhar, Mohamad 8.7<br />
Bader, David 2.23, 4.20, 4.21,<br />
4.26, 6.23<br />
Badia-Careaga, Claudio 7.7<br />
Bai, Yan 6.30<br />
Bakker, Martijn S5.5<br />
Bakkers, Jeroen S5.5, S8.1, 5.16,<br />
5.19<br />
Baldini, A 3.27<br />
Baldwin, H. Scott 5.21, 7.13, 8.12,<br />
8.24, S8.2<br />
Bamforth, Simon 6.19<br />
Banjo, Toshihiro 8.28<br />
Barnett, Joey 5.21, 8.12<br />
Barnett, Phil 1.23, 5.16, S5.5<br />
Barnette, Damien 8.17<br />
Barth, Jeremy 2.8<br />
Bartlett, Heather 1.25<br />
171<br />
Bartman, Thomas 8.29<br />
Barwick, Lee 8.21<br />
Bassat, Elad 2.9<br />
Baydoun, Serine 1.24<br />
Bayoumi, Ahmed 8.24<br />
Bean, Lora S1.5<br />
Behrens, Ann 4.36, 6.28<br />
Bellmann, Katherina S1.4<br />
Benham, Ashley 3.25<br />
Bennett, Mike 1.9<br />
Bentham, Jamie 6.19<br />
Beresian, Jean 1.24<br />
Berger, Felix S1.4<br />
Bergeron, Sarah 1.25<br />
Berry, James 8.16<br />
Bersell, Kevin 2.7<br />
Beyer, S 5.20<br />
Bezzina, Connie 5.16, S5.5<br />
Bhatia, Ankit S5.1<br />
Bhattacharya, Shoumo 6.19<br />
Bhattacharya, Subarna 3.20<br />
Bichell, David 8.24<br />
Bilardo, CM 1.23<br />
Bisgrove, Brent 3.14<br />
Bitar, FF S1.6, 1.24<br />
Black, Brian 4.33, 6.27<br />
Bodmer, Rolf S6.4<br />
Boskovski, Marko 6.11<br />
Bosman, Joshua 7.23<br />
Bosse, Kevin S8.6<br />
Boukens, Bas 5.15, 5.19<br />
Brade, Thomas 7.18<br />
Braganca, Jose 6.19<br />
Braitsch, Caitlin 5.9<br />
Brand, Thomas 5.29<br />
Bressan, Giorgio 8.11<br />
Bressan, Michael 5.11, 5.14, 5.17,<br />
S5.3<br />
Briggsa, Laura S8.5<br />
Brody, Matt 7.19<br />
Broka, Derrick 5.28<br />
Broman, Michael 5.12<br />
Bronson, Roderick S2.1<br />
Brothman, Arthur 6.8<br />
Broude, Eugenia 8.14, 8.25<br />
Brown, Chris S8.2<br />
Brown, Jesse 8.9<br />
Brudno, Michael 1.14<br />
Brueckner, Martina 6.11, 6.14, 6.18<br />
Bruneau, Benoit 1.11, 3.9, 4.33,<br />
S6.5, 7.9<br />
Brunelli, Luca 2.19<br />
Buac, Kristina S6.6<br />
Buck, Jessie S2.5<br />
Buckingham, Margaret S6.1<br />
Buikema, J 7.21<br />
Bunn, Rod 1.19, 4.18
Burchf, John S8.5<br />
Burkhard, Silja 5.19<br />
Burnicka-Turek, Ozanna S5.4, 3.10<br />
Burns, Caroline S7.2<br />
Burns, Ge<strong>of</strong>frey S7.2<br />
Burnside, Jessica 8.23<br />
Buske, Orion 1.14<br />
Butcher, Jonathan 4.35, 4.8<br />
Butzler, Matthew 6.9<br />
Camarata, Troy 3.26<br />
Camenisch, Todd 5.28<br />
Cameron, M 6.26<br />
Campione, Marina 5.18<br />
Cao, Jingjing 3.12<br />
Cao, Yunshan 2.11<br />
Caprioli, Richard 8.24<br />
Carlesso, Nadia S7.3<br />
Carnegie, Graeme 2.17<br />
Carrion, Katrina 8.20<br />
Casanova, Jesus 7.7, S7.4<br />
Cavallero, Susana 5.8, 7.18<br />
Chakraborty, Santanu 8.31<br />
Chang, Chien-fu 1.12, 1.21<br />
Chang, Sheng 7.10<br />
Chang, Wing 6.10<br />
Chatterjee, Bishwanath 1.10, 2.16<br />
Chédotal, Alain S4.3<br />
Chemtob, S 6.26<br />
Chen, Chiann-mun 6.19<br />
Chen, Dongying S4.2<br />
Chen, Guozhen 1.12, 1.17, 1.21<br />
Chen, Hanying S7.3<br />
Chen, Huijun 4.34<br />
Chen, Jau-Nian S5.6<br />
Chen, Jin-Lan 1.22<br />
Chen, Jinghai S2.4<br />
Chen, L 3.27<br />
Chen, Li 2.14<br />
Chen, Songhai S6.3<br />
Chen, Tsuey-Ming 3.19<br />
Chen, YiPing 8.23<br />
Cheng, Chia-ho S2.6<br />
Chhun, Danny 8.30<br />
Chi, Neil 6.27<br />
Chien, Kenneth S6.6<br />
Christine, Kathleen S4.5<br />
Christ<strong>of</strong>fels, Vincent S4.3, 5.15,5.16,<br />
5.19, 5.30, S5.5,<br />
S7.5<br />
Chuppa, Sandra 3.20<br />
Cieslik, Katarzyna 2.19<br />
Clark, Christopher 3.7<br />
Clarke, Jon S6.6<br />
Cleary, Michael S3.2<br />
Colan, Steven 2.7<br />
Colombo, Sophie S7.1<br />
Combs, Michelle 5.9<br />
Condic, Maureen 6.8<br />
172<br />
Conklin, Bruce 2.17<br />
Conlon, Frank S4.5, 7.25<br />
Constancia, Miguel 7.18<br />
Conway, Simon 4.13, S4.1, 4.29<br />
Cooley, Marion 3.7, 8.14, 8.25<br />
Cooney, Austin 6.7<br />
Coronel, Ruben 5.15<br />
Cosgrove, Catherine 6.19<br />
Cowan, Jason S1.3<br />
Cua, Clifford S1.5<br />
Czirok, Andras 6.17<br />
D'Amato, Gaetano S7.4<br />
D’Aniello, Enrico 6.16<br />
Damerla, Rama 1.10<br />
Dan, Kelvin 8.30<br />
DasLala, Tanmoy 2.7<br />
Davies, Benjamin 6.19<br />
Davis, Monica 6.29<br />
de la Pompa, José S7.4<br />
de Pater, Emma 5.19<br />
de Vlaming, Annemarieke S8.4, 8.18<br />
DeAngelis, Jessica 4.34<br />
Dees, Ellen 2.23, 3.24<br />
Degenhardt, Karl 4.22<br />
DeLaughter, Daniel 5.21, 8.12<br />
Demarest, Bradley 3.14, 6.8<br />
Deshmukh, V. 3.13<br />
Deshwar, Ashish 6.24<br />
Dettman, Robert S8.5, 8.10<br />
Devasthali, Vidusha S3.2<br />
Devine, Patrick 3.9, 4.33, S6.5<br />
Devine, William 1.17<br />
Dickinson, Mary 4.10<br />
Dobarro, David S7.4<br />
Doe, Chris S3.2<br />
Doevendans, P 7.21<br />
Dohn, TE 6.20<br />
Dollé, Pascal 4.16, S7.6<br />
Domian, I 7.21<br />
Domínguez, Jorge 5.13, S7.5<br />
Dooley, Kenneth S1.5<br />
Dorfmeister, K 4.25<br />
Dorn, Cornelia S1.4<br />
Dorn, Michael 4.24<br />
Dorr, Kerry 7.25<br />
dosRemedios, Cris 2.7<br />
Doughman, Yong 1.7, 2.18, 8.21<br />
Doughman, Yongqiu 5.23<br />
Dressler, Gregory 7.9<br />
Drysdale, T 7.12<br />
Duan, Dongsheng 2.13<br />
Dubois, Nicole 1.8<br />
Duester, Gregg 7.18<br />
Dufour, Alexandre S6.1<br />
Dunkel, Ilona S1.4<br />
Dunlevy, Louisa 1.9<br />
Dupays, Laurent 3.28<br />
Dupuis, Loren S8.3
Dvorak, Katarina 8.9<br />
Dyer, Laura 5.7<br />
Dyo, Jeffrey 8.20<br />
Earley, Judy 3.8, S5.1, 8.15<br />
El-Rassy, IG S1.6<br />
Ellis, James 6.10<br />
Encinas, Alejandra 8.7<br />
Engel, Felix S4.6<br />
Enomoto, Hirokazu 1.26, S3.3<br />
Epstein, Jonathan 4.22<br />
Espana, Karina S1.5<br />
Evans-Anderson, Heather 2.8<br />
Evans, Todd 3.21<br />
Fahed, AC S1.6<br />
Fahrenbach, John S5.4<br />
Fang, Yi 2.10<br />
Farin, Henner 5.22<br />
Farkas, Deborah 1.12, 1.15, 1.17,<br />
1.22, 2.16<br />
Fatakia, Sarosh 1.10, 1.12<br />
Faul, Christian 8.26<br />
Feingold, Eleanor S1.5<br />
Feng, Qingping 5.27<br />
Fernández-Friera, Leticia S7.4<br />
Finnell, Richard 4.16, S7.6<br />
Fisher, Elizabeth 1.9<br />
Fisher, Steven 2.15<br />
Fishman, Glenn 3.21<br />
Fitzpatrick, David 1.13<br />
Flagg, Alleda 5.25<br />
Foker, John 8.16<br />
Francey, Lauren 4.22<br />
Francis, Richard 1.12, 1.15, 1.17,<br />
1.20, 1.22<br />
Franco, Diego 5.13<br />
Franklin, Sarah S5.6<br />
Fresco, Victor 3.7, 8.14, 8.25<br />
Friedland-Little, Joshua 3.10<br />
Fukuda, Keiichi 1.26, S3.3<br />
Fulcoli, FG 3.27<br />
Furukawa, Tetsushi 1.26, S3.4<br />
Gallegos, Thomas 8.20<br />
Gao, Zhiguang 3.8, S5.1<br />
García-Pavía, Pablo S7.4<br />
Garg, Vidu 7.10, S8.6<br />
Garrec, Jean-François S6.1<br />
Garry, Daniel 4.36, 6.28<br />
Gasch, Alexander 1.26<br />
Gay, Leslie S3.2<br />
Ge, Kai 7.9<br />
Geiger, Benjamin 2.9<br />
George, Vanessa S7.1<br />
Geyer, Stefan 4.23, 4.24, 4.25<br />
Ghatak, S 8.13, 8.19<br />
Gimeno, Juan S7.4<br />
Glenn, Nikki 8.29<br />
Gokey, Jason 6.13<br />
Goldhaber, Joshua S5.6<br />
173<br />
Gonzalez-Rajal, Álvaro S7.4<br />
Gordillo, Miriam 3.21<br />
Gorsi, Bushra 3.14, S3.1<br />
Graham, Dionne 2.7<br />
Greene, Stephanie 3.17<br />
Greulich, Franziska 5.22<br />
Grieskamp, Thomas 5.30<br />
Grover, S 7.12<br />
Gruber, Peter 4.22<br />
Grunert, Marcel S1.4<br />
Gu, Shi 1.7, 2.18, 8.21<br />
Guggilam, Anuradha S8.6<br />
Gundry, Rebekah 3.20<br />
Guner-Ataman, Burcu S7.2<br />
Gunst, Quinn 5.15<br />
Gupta, Vikas 2.10, S6.2<br />
Guzman, Claudia S2.6<br />
Haaning, Allison 6.12<br />
Hacker, Timothy 7.19<br />
Hajdu, Zoltan 8.22<br />
Halabi, R 7.12<br />
Hammond, Harriet 8.8<br />
Han, Mingda 1.20<br />
Hanna, Alex 4.19<br />
Hans, Chetan S8.6<br />
Harikrishnan, Keerthi 8.14, 8.25<br />
Harmelink, Cristina 7.15<br />
Harmon, Andrew 4.29<br />
Harvey, Brian 8.16<br />
Harvey, Richard 3.28, 4.29, 6.28<br />
Hascall, VC 8.19<br />
Hattori, Fumiyuki 1.26<br />
Hatzopoulos, Antonis 3.18<br />
He, Aibin 3.12<br />
He, Bin 4.32<br />
Heideman, Warren 5.24, 5.26<br />
Hetzer, Roland S1.4<br />
Higashikuse, Yuta 1.26<br />
Hill, Cyndi 5.21<br />
Hill, Jonathon 3.14, 6.8<br />
Hinek, Aleksander 1.8, 6.10<br />
Hinton, Robert S8.3, 8.7, 8.8, 8.11<br />
Hoen, Peter S5.5, 5.16<br />
H<strong>of</strong>fman, S 7.17,8.19<br />
H<strong>of</strong>fmann, Andrew 3.10, 7.23<br />
H<strong>of</strong>schreuder, Niels S7.5<br />
H<strong>of</strong>steen, Peter 5.24, 5.26<br />
Hogan, Benjamin 4.34<br />
Holdway, Jennifer 2.10<br />
Hollands, Andrew 8.20<br />
Holtrup, Brandon 3.26, 8.10<br />
Holtz, Mary 5.10<br />
Hom, Jennifer 2.20<br />
Horton, Anthony 3.7<br />
Hou, Shuan 4.17<br />
Hou, Shuan-Yu 7.14<br />
Hsiao, Edward 2.17<br />
Hsu, Pearl 6.9
Hu, Jiangyong 3.18<br />
Huang, Can 1.22<br />
Huang, Hannah 7.10<br />
Huang, Jie S5.6<br />
Huang, Nianyuan S8.6<br />
Huang, Ying 2.22<br />
Huang, Yu 2.17<br />
Huang, Zhan-Peng S2.4<br />
Hubmacher, Dirk S1.1<br />
Huk, Danielle 8.8<br />
Hutson, Mary 4.14<br />
Iacovino, Michelina 6.28<br />
Ibañez, Borja S7.4<br />
Igarashi, Takashi 4.31<br />
Ilgun, A 1.23<br />
Illingworth, EA 3.27<br />
Imanaka-Yoshida, Kyoko 4.27<br />
Inai, Kei 8.23<br />
Islas, Jose 3.19, 3.25<br />
Ito, Aiko 4.19<br />
Iyer, Dinakar 3.19<br />
Izquierdo-García, José S7.4<br />
Jay, Patrick S1.2<br />
Jenkins, Michael 1.7, 2.18, 8.21<br />
Jerde, Audrey 3.8, 8.15<br />
Jiao, Kai 7.15<br />
Jiménez-Borreguero, Luis S7.4<br />
John, Manorama 6.9<br />
Jungblut, Benno S4.6<br />
Kaartinen, Vesa 7.20<br />
Kageyama, Toshimi 1.26<br />
Kakarla, Jayant 7.22<br />
Kamar, Amina 1.24<br />
Kamide, Christine 4.19<br />
Kamp, Timothy 6.9<br />
Kaneda, Ruri 1.26, S3.3<br />
Kang, Bo 4.32<br />
Kang, Guoxin 3.21<br />
Kang, Jione 6.27<br />
Karra, Ravi 2.10<br />
Karunamuni, Ganga 1.7<br />
Kawakami, Atsushi 1.26<br />
Keavney, Bernard 6.19<br />
Keith, Kimberly S2.1<br />
Keller, Gordon 1.8, 6.10<br />
Kelly, Robert 5.20, S7.5<br />
Kenchegowda, Doreswamy 2.15<br />
Kennedy, Karen 6.10<br />
Kern, Christine 8.14, 8.25, S8.3<br />
Keyte, Anna 4.14<br />
Khattak, Shahryar 6.10<br />
Khokha, Mustafa 6.11, 6.14<br />
Kida, Yasuyuki 8.28<br />
Kidokoro, Hinako 6.15<br />
Kim, Andrew 1.12, 1.17, 1.21<br />
Kim, Andy 2.16<br />
Kim, Chul 3.23<br />
Kim, Eun 2.21<br />
174<br />
Kim, Gene S5.1, S5.4, 5.12<br />
Kim, Jieun 2.22<br />
Kim, Ki-Sung S4.4, 4.31, 6.22<br />
Kim, Kwangho 3.18<br />
Kim, Youngsil 1.12, 1.15, 1.21<br />
Kimura, Akinori 1.26<br />
Kimura, Taizo 4.27<br />
King, Isabelle S3.6<br />
Kinnear, Caroline 6.10<br />
Kirby, Margaret 4.14, 4.15<br />
Kispert, Andreas S5.2, 5.22, 5.30<br />
Klaassen, Sabine S1.4<br />
Klimek, Margaret 8.26<br />
Klotz, Linda 4.30<br />
Kodo, Kazuki 4.12<br />
Koenig, Sara S8.6<br />
Kohli, Vikram 4.9, 6.27, 8.29<br />
Kojima, Mizuyo S3.4<br />
Kolander, Kurt 5.10<br />
Kong, Young-Y S7.4<br />
Kontaridis, Maria S2.1<br />
Koo, Hyun-Young 4.19<br />
Kosaka, Yasuhiro 2.19<br />
Koshiba-Takeuchi, Kazuko S3.4, 3.9, 3.22<br />
Kotecha, Surendra 3.28<br />
Kotton, Darrell 1.8<br />
Krantz, Ian 4.22<br />
Krcmery, Jennifer 8.10<br />
Krug, Edward 6.29, 7.17<br />
Kudo, Akira 1.26<br />
Kühn, Bernhard 2.7<br />
Kume, Tsutomu 4.19<br />
Kurihara, Hiroki S4.4, 4.31, 6.22<br />
Kurihara, Yukiko S4.4, 4.31, 6.22<br />
Kurokawa, J. 3.22<br />
Kuyumcu-Martinez M. N. 3.13<br />
Kweon, Junghun 7.23<br />
Kwon, C. 3.22<br />
Kwon, Ohyun S5.6<br />
Kyba, Michael 6.28<br />
Labeit, Siegfried 1.26<br />
Labiner, Hanna 7.25<br />
Labosky, Patricia 4.26<br />
Lagendijk, A. S8.1<br />
Lajiness, Jacquelyn 4.13<br />
Lana-Elola, Eva 1.9<br />
Lansford, Rusty 6.17<br />
Lasheras, Juan 8.20<br />
Laura, Briggs 7.22<br />
Lauriol, Jessica S2.1<br />
Le Saux, Claude 6.29<br />
Leatherbury, Linda 1.12, 1.15, 1.17,<br />
1.21<br />
Leclerc, S 6.26<br />
Lee, Ethan 3.18<br />
Lee, Ji-Eun 7.9<br />
Lee, Kyu-Ho S2.1, 3.7<br />
Lee, Mary 8.31
Lee, Youngsook 7.19<br />
Lencinas, Alejandro 8.30<br />
Leung, Carmen 5.27<br />
Levine, Robert S8.4, 8.18<br />
Lezin, George 2.19<br />
Li, Dejia 2.13<br />
Li, Jun 4.22<br />
Li, Li 4.22<br />
Li, Peng 5.8, 7.18<br />
Li, Sean 3.11<br />
Li, Xinli 2.11<br />
Li, You 1.10, 1.12, 1.15,<br />
2.16<br />
Liang, Dong 7.14<br />
Liberko, Kevin 6.9<br />
Lien, Ching-Ling 2.22<br />
Lilly, Brenda S8.6<br />
Lin, Christopher 6.18<br />
Lin, Fang S6.3<br />
Lin, Song-Chang 4.16<br />
Lin, Yi-Fan 7.8<br />
Lin, Zhiqiang S2.5<br />
Linask, Kersti 1.7, 1.20<br />
Lincoln, Joy S8.6, 8.8, 8.17,<br />
8.26<br />
Lindsey, Stephanie 4.8, 4.35<br />
Little, Charles 6.17<br />
Liu, Fang S5.4<br />
Liu, Gary S5.3, 5.11<br />
Liu, Hongbin 2.15<br />
Liu, Jiandong S6.4<br />
Liu, Murong 5.27<br />
Liu, Ting 4.19<br />
Liu, Ting-Chun 3.21<br />
Liu, Xiao 4.18<br />
Liu, Xiaoqian 4.29<br />
Liu, Xiaoqin 1.12, 1.17, 1.21,<br />
2.16<br />
Liu, Yu 3.19, 3.25, 6.7<br />
Liu, Zhao 1.7<br />
Liu, Zhi-Ping S2.2<br />
Lo, Cecilia 1.10, 1.11, 1.12,<br />
1.15, 1.17, 1.19,<br />
1.21, 2.16, 4.18<br />
Locke, Adam S1.5<br />
Lockhart, Marie 7.11, S8.5<br />
Lohr, Jamie 6.28<br />
Losordo, Douglas 4.19<br />
Louie, Jonathan 5.14<br />
Lu, Jia 3.21<br />
Lu, Kui S5.6<br />
Lu, Xiangru 5.27<br />
Lucchesi, Pamela S8.6<br />
Ludwig, Andreas S6.6<br />
Luxán, Guillermo S7.4<br />
Lynn, Heather 1.12, 1.15, 1.21<br />
Lyons, Gary 6.9<br />
Ma, Qing 3.12<br />
175<br />
Ma, Yanlin 2.14<br />
Mably, John 4.28<br />
MacGrogan, Donal S7.4<br />
Maeda, Kazuhiro S4.4<br />
Maguire, Colin 6.8<br />
Mahmut, Naila 6.10<br />
Makino, Shinji 1.26, S3.3<br />
Makova, Svetlana 6.11<br />
Malloy, Lindsey 1.25<br />
Maloney, David S2.2<br />
Manickaraj, Ashok 1.14<br />
Mannens, MMAM 1.23<br />
Markwald, Roger 7.17, S8.4, 8.13,<br />
8.18, 8.19, 8.22,<br />
8.23<br />
Marnett, Larry 3.18<br />
Martens, Spencer 3.8<br />
Martin, Cindy 4.36, 6.28<br />
Martin, James 3.17, 6.30, 8.32<br />
Martínez-Poveda, Beatriz S7.4<br />
Martinez, Fernando S7.4<br />
Martucciello, S 3.27<br />
Marx, Ken S2.6<br />
Maslen, Cheryl S1.5, 1.16<br />
Massera, Daniele 4.22<br />
Maurer, B 4.23, 4.24, 4.25<br />
Mayer, John 8.24<br />
McKane, Melissa 1.25, 8.29<br />
McNally, Elizabeth S5.4<br />
McSharry, Saoirse S5.1, 5.12, 8.15<br />
Mebus, Siegrun S1.4<br />
Medrano, Constancio S7.4<br />
Meilhac, Sigolène S6.1<br />
Mercer, Catherine 1.13<br />
Mercer, Sarah S2.6<br />
Mercola, Mark 8.23<br />
Merks, R S8.1<br />
Merryman, David S8.2<br />
Mesbah, Karim S7.5<br />
Mettler, Brett 8.24<br />
Michalik, Liliane 2.19<br />
Mikawa, Takashi S5.3, 5.11, 5.14,<br />
5.17<br />
Mikkola, Hanna 4.29<br />
Miller, Michael S3.2<br />
Miller, Paul 2.23<br />
Miquerol, L 5.20<br />
Mironova, Ekaterina 3.24<br />
Misener, Sol 4.19<br />
Mishina, Yuji 8.23<br />
Misra, Chaitali 7.10<br />
Misra, Ravi 5.10<br />
Misra, S 8.13, 8.19<br />
Mital, Seema 1.14, 1.8, 6.10<br />
Miura, Grant 6.25<br />
Miyagawa-Tomita, Sachiko S4.4, 6.22<br />
Miyasaka, Kota 8.28<br />
Mjaatvedt, Corey 6.29
Mohun, Timothy 1.9, 3.28<br />
Molero, Magdalena 5.13<br />
Mollova, Mariya 2.7<br />
Mommersteeg, Mathilda S4.3<br />
Montserrat, Lorenzo S7.4<br />
Moon, Anne 7.23<br />
Mooney, Sean S1.5<br />
Moorman, Antoon 1.23, 5.15, 5.30,<br />
S7.5<br />
Moreno-Rodriguez, RA 7.17, 8.19<br />
Morikawa, Yuka 8.32<br />
Morita, Y. 3.22<br />
Moskowitz, Ivan 3.10, 3.23, S5.4,<br />
7.23<br />
Mukherjee, Amrita 3.18<br />
Mukouyama, Y. 4.7<br />
Munjal, Charu 8.11<br />
Murata, Mitsushige 1.26<br />
Nagy-Mehesz, Agnes 8.22<br />
Nakamaura, Ryo S3.4<br />
Nakano, Atsushi 4.29<br />
Nakano, Haruko 4.29<br />
Nakashima, Yasuhiro 4.29<br />
Nasreen, Shampa 3.24<br />
Nassano-Miller, Carey 4.19<br />
Navetta, Alicia 5.17<br />
Neeb, Zachary S4.1<br />
Neel, Benjamin 1.8<br />
Nelson, Daryl 6.9<br />
Nemer, Georges 1.24<br />
Nemer, GM S1.6<br />
Nevis, Kathleen S7.2<br />
Ni, Terri 3.18<br />
Niederreither, Karen 4.16, S7.6<br />
Nigam, Sanjay 8.20<br />
Nigam, Vishal 8.20<br />
Nikolic, Iva S4.6<br />
Nishimura, Nozomi 4.8, 4.35<br />
Nishinakamura, R. 3.22<br />
Nishiyama, Koichi S4.4, 4.31<br />
Nizet, Victor 8.20<br />
Nobrega, Marcelo S3.5, S5.4<br />
Norden, Julia S5.2, 5.30<br />
Norris, Russel 7.11, S8.4, S8.5,<br />
8.13, 8.18, 8.19,<br />
8.22<br />
O’Kelly, Ita 1.13<br />
Oakey, Rebecca 7.13<br />
Oda, Mayumi 1.26, S3.3<br />
Odelberg, Shannon S2.6<br />
Ogawa, Hisao S4.4<br />
Ogura, Toshihiko 8.28<br />
Okabe, Masataka 6.15<br />
Olivey, Harold 5.12, 5.25<br />
Olivo-Marin, Jean-Christophe S6.1<br />
Omi, Minoru 8.28<br />
Oostra, RJ 1.23<br />
Opoka, Amy 8.11<br />
176<br />
Paffett-Lugassy, Noelle S7.2<br />
Pajkrt, E 1.23<br />
Palencia-Desai, Sharina 4.9, 6.27<br />
Palmer, James 6.8<br />
Papaioannou, Virginia S7.5<br />
Pardhanani, Pooja 2.8<br />
Park, Shin-Young 2.7<br />
Parnavelas, John S4.3<br />
Paschaki, Marie 4.16<br />
Pashmforoush, Mohammad 4.29<br />
Pastuszko, Peter 8.20<br />
Patel, Vickas S5.4<br />
Patel, Vishal 8.20<br />
Paterson, Scott 4.34<br />
Patra, Chinmoy S4.6<br />
Patterson, Cam 5.7<br />
Pegram, Kelly 4.14<br />
Pekkan, Kerem 4.18<br />
Perez-Pomaresd, Jose S8.5<br />
Petchey, Louisa S2.3<br />
Peterson, Lindsy 1.7, 2.18, 8.21<br />
Peterson, Richard 5.24, 5.26<br />
Pfaltzgraff, Elise 4.26<br />
Phelps, Aimee 7.11, 7.22, S8.5<br />
Phillips, Amanda 4.22<br />
Pierick, Alyson 1.25<br />
Pizarro, Gonzalo S7.4<br />
Plavicki, Jess 5.24, 5.26<br />
Polk, Renita 1.18<br />
Pop, Sorin S6.1<br />
Porter, George 2.20<br />
Poss, Kenneth 2.10, S6.2<br />
Postma, AV 1.23<br />
Potaman, Vladimir 3.19, 3.25<br />
Powers, Pat 6.9<br />
Prados, Belén S7.4<br />
Prejusa, Allan 3.19<br />
Prickett, Adam 7.13<br />
Pu, William S2.5, 3.12, 7.10<br />
Pyles, Lee 8.16<br />
Qian, Ling 2.14<br />
Qu, Xianghu S8.2<br />
Quaggin, Susan 5.9<br />
Quinn, Malgorzata 6.12<br />
Ragni, Chiara S6.1<br />
Rahmani, Mahdis 8.32<br />
Rainato, Giulia 5.18<br />
Rajchman, Dana 2.9<br />
Ramchandran, Ramani 3.16<br />
Ramirez-Bergeron, Diana 5.23<br />
Rana, Sameer S7.5<br />
Reese, Jeff 4.26<br />
Reeves, Roger S1.5, 1.18<br />
Regmi, Suk S1.2<br />
Rehn, Kira 4.9<br />
Reidler, Andy 1.19<br />
Rellinger, Eric 3.18<br />
Ren, Yi 4.36, 6.28
Reshey, Benjamin S1.5<br />
Reyes, L 7.17<br />
Rhinn, Muriel S7.6<br />
Ricciardi, Filomena S4.6<br />
Riley, Paul S2.3<br />
Robinson, Jamille 8.12<br />
Robson, Andrew 6.14<br />
Rollins, Andrew 1.17, 2.18, 8.21<br />
Rongish, Brenda 6.17<br />
Rosen, Jonathan 4.28<br />
Ross, Kristen 8.30<br />
Rubenstein, Peter 1.25, 8.29<br />
Rudat, Carsten S5.2<br />
Runyan, Raymond 8.9, 8.30<br />
Rydeen, Ariel 7.16<br />
Saadeh, Heba 7.13<br />
Sabater-Molina, María S7.4<br />
Sadek, Hesham S3.4<br />
Sadleir, Rudyard 8.10<br />
Sahara, Makoto S6.6<br />
Saijoh, Yukio 6.15<br />
Sakabe, Noboru S3.5<br />
Salem, Elie 1.24<br />
Salomonis, Nathan 2.17<br />
Sano, Motoaki 1.26<br />
Sanz-Ezquerro, Juan 7.7<br />
Sarig, Rachel 2.9<br />
Sasano, Tetsuo S3.4<br />
Sasidharan, Rajkumar 4.29<br />
Sato, Takahiro 4.31<br />
Sauls, Kimberly S8.4, S8.5, 8.18<br />
Savla, Jainy 2.7<br />
Schachterle, William 4.33<br />
Schaffer, Chris 4.8, 4.35<br />
Schell, Thomas S7.1<br />
Schlesinger, Jenny S1.4<br />
Schlueter, Jan 5.29<br />
Schmidt, Mirko S4.6<br />
Schneider, Jürgen 6.19<br />
Schoen, Kevin 6.9<br />
Schoenwolf, Gary 6.15<br />
Schramm, Charlene 1.11<br />
Schredelseker, Johann S5.6<br />
Schueler, Markus S1.4<br />
Schulkey, Claire S1.2<br />
Schulz, Reiner 7.13<br />
Schupp, Marcus-Oliver 3.16<br />
Schuster-Gossler, Karin 5.22<br />
Schwacke, John 6.29<br />
Schwartz, Robert 2.14, 2.21, 3.19,<br />
3.25, 4.29, 6.7<br />
Scott, Ian 6.24<br />
Scott, Mark 2.17<br />
Sedmera, D 5.20<br />
Seidman, CE S1.6<br />
Seidman, J S1.6, 1.11, 5.21,<br />
8.12<br />
Seok, Hee S2.4<br />
177<br />
Sewell, M-K S8.2<br />
Shehane, Richard 6.25<br />
Shekhar, Akshay 4.8, 4.35<br />
Shen, Hua 5.8, 7.18<br />
Sherman, Stephanie S1.5<br />
Shibbani, Kamel 1.24<br />
Shim, Kyoo 4.19<br />
Shimizu, Hirohito S5.6<br />
Shin, Jae-Ho 4.29<br />
Shou, Weinian S7.3<br />
Shung, Wendy 1.12, 1.17, 1.19,<br />
1.21<br />
Silberstein, Leslie 2.7<br />
Simeonov, Anton S2.2<br />
Simon, Hans-Georg S2.6, 3.26, 8.10<br />
Singh, Manvendra 4.22<br />
Sinha, Tanvi 7.24<br />
Slaugenhaupt, Susan S8.4, 8.18<br />
Slender, Amy 1.9<br />
Sluijter, J 7.21<br />
Smemo, Scott S3.5, S5.4<br />
Smith, Kelly 4.34<br />
Snider, Paige S4.1, 4.13<br />
Sogah, Vanessa 4.28<br />
Solnica-Krezel, Lilianna S7.1<br />
Soubra, Ayman 1.24<br />
Später, Daniela S6.6<br />
Sperling, Silke S1.4<br />
Spicer, Doug 8.31<br />
Spindler, Matthew 2.17<br />
Srinivasan, Ashok 1.10, 1.12, 2.16<br />
Srivastava, Deepak 2.17<br />
Stachura, David 6.25<br />
Stadt, Harriett 4.14<br />
Stainier, Didier 4.33<br />
Stanford, William 6.10<br />
Stankunas, Kryn S3.2<br />
Stephens, Lauren 3.18<br />
Su, Yan 8.24<br />
Sucov, Henry 5.8, 7.18<br />
Sugi, Yukiko 8.19, 8.23<br />
Sugizaki, H. 3.22<br />
Sumanas, Saulius 4.9, 6.27, 8.29<br />
Sutherland, Mardi 6.12<br />
Suzuki, Takeshi 1.26<br />
Svensson, Eric 3.8, S5.1, 5.12,<br />
5.25, 8.15<br />
Swinburne, Ian 7.8<br />
Sylva, Marc 1.23, 5.15<br />
Szabo, A S8.1<br />
Szeto, Kai 8.20<br />
Szmacinski, Marta S4.5<br />
Taketo, Makoto S5.2<br />
Takeuchi, Jun S3.4, 3.22<br />
Tamura, Koji 6.15<br />
Tan, Zhi-Ping 1.22<br />
Tanos, RC S1.6<br />
Tao, Ge S8.6
Tao, Jiayi 5.23<br />
Targ<strong>of</strong>f, Kimara S7.1<br />
Tariq, Muhammad S1.3<br />
Tavares, Andre 8.9<br />
Taylor, Joan S4.5<br />
Temple, Sally S3.2<br />
Tennstedt, Sharon 1.11<br />
Tessadori, Federico S5.5, 5.16, 5.19<br />
Tevosian, Sergei 7.19<br />
Thomas, Penny 7.20<br />
Thomason, Rebecca 4.21, 6.23<br />
Thompson, Heather S3.2<br />
Thompson, Tadeo 6.10<br />
Thorne, Cutris 3.18<br />
Thusberg, Janita S1.5<br />
Tiburcy, Malte 1.8<br />
Timmermann, Bernd S1.4<br />
Tobita, Kimimasa 1.12, 1.15, 1.17,<br />
1.21<br />
Togi, Kyonori 3.9<br />
Torroja, Carlos S7.4<br />
Towers, Norma 3.28<br />
Townsend, Todd 3.15<br />
Traver, David 6.25<br />
Trzebiatowski, Joshua 6.9<br />
Tsuchihashi, Takatoshi 4.12<br />
Tsukahara, Yuko S3.4, 3.22<br />
Turbendian, Harma 3.21<br />
Twal, Waleed 3.7, 8.14, 8.25<br />
Tybulewicz, Victor 1.9<br />
Tzahor, Eldad 2.9<br />
Uchida, Keiko 4.12<br />
Uchijima, Yasunobu S4.4, 4.31<br />
Udan, Ryan 4.10<br />
Uribe, Veronica 7.7<br />
Vadakkan, Tegy 4.10<br />
van den Boogaard, Malou S5.5, 5.16<br />
van den H<strong>of</strong>f, Maurice 5.15, 7.22, S8.5<br />
van Handel, Ben 4.29<br />
van Maarle, MC 1.23<br />
van Rijn, RR 1.23<br />
van Weerd, Henk 5.19<br />
van Wijkb, Bram S8.5<br />
Vanderpool, Nicole 1.25<br />
Ventura, Brit S3.2<br />
Verkerk, Arie 5.19<br />
Verma, S. K. 3.13<br />
Violette, Kate S8.2<br />
Visconti, Richard 8.22<br />
Vogler, Georg S6.4<br />
von Gise, Alexander S2.5, 3.12<br />
Vondriska, Thomas S5.6<br />
Vue, Zer S3.2<br />
Wahli, Walter 2.19<br />
Wahlig, Taylor 2.10<br />
Walsh, Stuart 2.7<br />
Wang, Bing 7.24<br />
Wang, Da-Zhi S2.4<br />
178<br />
Wang, Jianbo 7.24<br />
Wang, JingYing 3.11<br />
Wang, Jinhu 2.10<br />
Wang, Jun 2.14, 2.21, 3.17<br />
Wang, Kevin S5.6<br />
Wang, Lauren S1.1<br />
Wang, Liyin 1.12, 1.15<br />
Wang, Qiaohong 4.22<br />
Wang, Shuyun 6.12<br />
Wang, Xia 4.17<br />
Wang, Zhinong 4.32<br />
Ware, Stephanie S1.3, 6.12<br />
Wassilew, Katharina S1.4<br />
Watanabe, Michiko 1.7, 2.18, 5.23,<br />
8.21<br />
Watkins, Hugh 6.19<br />
Watson-Scales, Sheona 1.9<br />
Waxman, Joshua 6.16, 6.20, 7.16<br />
Wedemeyer, Elesa 1.25<br />
Wen, Kuo-Kuang 1.25, 8.29<br />
Weng, Kuo-Chan 3.19, 3.25<br />
Weninger, Wolfgang 4.23, 4.24, 4.25<br />
Wessels, Andy 7.11, 7.22, S8.4,<br />
S8.5, 8.18<br />
Whitehead, Kevin 2.19<br />
Wicking, Carol 4.34<br />
Wiedemeier, Olena 3.20<br />
Williams, Katherine S8.4, 8.18<br />
Willing, Erin 6.9<br />
Wilson, David 1.13<br />
Wilson, Robert 3.28<br />
Winters, Nichelle 4.20, 6.23<br />
Winters, Niki 4.21<br />
Wirrig, Elaine 7.11<br />
Wong, Elaine S5.5, 5.16<br />
Wong, Li 1.21<br />
Wu, Xiangqi 2.11<br />
Wu, Xiushan 8.27<br />
Wylie, John 4.33<br />
Wythe, Joshua 3.9, 4.33, S6.5<br />
Xiao, Jian 4.32<br />
Xiao, Qi 4.11<br />
Xie, Bing 4.32<br />
Xie, Linglin 3.10, 3.29<br />
Xie, Shuying 3.18<br />
Xu, Xueping 6.7<br />
Yagi, Hisato 1.10, 2.16<br />
Yahalom, Yfat 2.9<br />
Yalcin, Huseyin 4.8, 4.35<br />
Yamagishi, Hiroyuki 4.12<br />
Yang, Holly 7.23<br />
Yang, Jin-Fu 1.22<br />
Yang, Ke 5.23<br />
Yang, Litao 3.25<br />
Yang, PoAn 5.17<br />
Yang, Yi-Feng 1.22<br />
Yang, Zhongzhou 2.11, 2.12, 4.11<br />
Yap, Choon 4.18
Ye, Ding S6.3<br />
Ye, Li 4.28<br />
Yehya, Amin 1.24<br />
Yelon, Deborah 6.25, S7.1, 7.8<br />
Yoder, Mervin S7.3<br />
Yoon, Sung 1.26<br />
Yoshida, Toshimichi 4.27<br />
Yoshimoto, Momoko S7.3<br />
Yoshimura, Koichi 4.27<br />
Yost, H. Joseph 1.11, 2.19, S3.1,<br />
3.14, 3.15, 6.8<br />
Yu, Wei 2.14, 2.21<br />
Yuan, Baiyin 2.12<br />
Yuasa, Shinsuke 1.26, S3.3<br />
Yue, Yongping 2.13<br />
Yutzey, Katherine 5.9, 8.31<br />
Zendron, Birgit 4.23<br />
Zhang, Boding S2.1, 3.7<br />
179<br />
Zhang, Chunxian 3.24<br />
Zhang, Jin S7.3<br />
Zhang, Ke 3.10<br />
Zhang, Kurt 3.29<br />
Zhang, Lifeng 1.20<br />
Zhang, Qing-Jun S2.2<br />
Zhang, Wei-Zhi 1.22<br />
Zhang, Wenjun S7.3<br />
Zhang, Yufeng 4.32<br />
Zhao, Ning S8.6<br />
Zhong, Tao 3.18<br />
Zhou, Bin S8.2, 8.23<br />
Zhou, Pingzhu S2.5, 3.12<br />
Zhou, Sherry 6.18<br />
Zierold, Claudia 4.36, 6.28<br />
Zimmermann, Wolfram 1.8<br />
Zong, Hui S3.2
Registrants<br />
Radwan Abu-Issa<br />
Department <strong>of</strong> Natural Sciences<br />
<strong>University</strong> <strong>of</strong> Michigan-Dearborn<br />
4901 Evergreen Road<br />
Dearborn, Michigan, 48128<br />
United States<br />
rabuissa@umich.edu<br />
Molly Ahrens<br />
Department <strong>of</strong> Pediatrics<br />
Children's Memorial Research Center<br />
2430 North Halsted<br />
<strong>Chicago</strong>, IL, 60614<br />
United States<br />
mollyjahrens@u.northwestern.edu<br />
Anastasiia Aleksandrova<br />
Department <strong>of</strong> Anatomy and Cell<br />
Biology<br />
<strong>University</strong> <strong>of</strong> Kansas Medical Center<br />
3901 Rainbow Blvd<br />
Kansas City, KS, 66103<br />
United States<br />
aaleksandrova@kumc.edu<br />
Christina Alfieri<br />
Department <strong>of</strong> MCB<br />
CCHMC<br />
240 Albert Sabin Way<br />
Cincinnati, OH, 45229<br />
United States<br />
alsyp6@cchmc.org<br />
Patrick Allison<br />
Department <strong>of</strong> Pharmacology and<br />
Toxicology<br />
<strong>University</strong> <strong>of</strong> Arizona College <strong>of</strong><br />
Pharmacy<br />
1703 East Mabel<br />
Tucson, AZ, 85721<br />
United States<br />
pallison@pharmacy.arizona.edu<br />
Martha Alonzo-Johnsen<br />
Department <strong>of</strong> Biology Department<br />
Duke <strong>University</strong> Medical Center<br />
Research Drive - Jones Building 402<br />
Durham, NC, 27710<br />
United States<br />
mra12@duke.edu<br />
Jeffrey Amack<br />
Department <strong>of</strong> Cell and Developmental<br />
Biology<br />
SUNY Upstate Medical <strong>University</strong><br />
309 Weiskotten Hall<br />
750 East Adams Street<br />
Syracuse , New York, 13210<br />
United States<br />
amackj@upstate.edu<br />
Nicholas Andersen<br />
Neonatal Perinatal Research Institute,<br />
Department <strong>of</strong> Pediatrics<br />
Duke <strong>University</strong> Medical Center<br />
102 Research Drive<br />
Jones Building Room 402<br />
Durham, NC, 27710<br />
United States<br />
Nicholas.Andersen@duke.edu<br />
SiangYun Ang<br />
Department <strong>of</strong> Biomedical Sciences<br />
Gladstone Institute <strong>of</strong> Cardiovascular<br />
Disease<br />
1650 Owens St<br />
San Francisco, CA, 94158<br />
United States<br />
yun.ang@gladstone.ucsf.edu<br />
Peggi Angel<br />
Department <strong>of</strong> Biochemistry<br />
Vanderbilt <strong>University</strong><br />
465 21st Ave S<br />
Room 9160 Bldg MRB3<br />
Nashville, TN, 37072<br />
United States<br />
peggi.angel@vanderbilt.edu<br />
Toshiyuki Araki<br />
Department <strong>of</strong> Stem cell &<br />
Developmental biology<br />
Ontario Cancer Institute<br />
101 College street TMDT8-355<br />
Toronto, ON, M5G1L7<br />
Canada<br />
taraki@uhnres.utoronto.ca<br />
Yuichiro Arima<br />
Department <strong>of</strong> Physiological Chemistry<br />
and Metabolism, Graduate School <strong>of</strong><br />
Medicine<br />
The <strong>University</strong> <strong>of</strong> Tokyo<br />
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-<br />
0033, Japan<br />
ayuichiro@hotmail.com<br />
David Arnolds<br />
Department <strong>of</strong> Pediatrics<br />
<strong>University</strong> <strong>of</strong> <strong>Chicago</strong><br />
900 E 57th St.<br />
KCBD 5240 LB-1<br />
<strong>Chicago</strong>, IL, 60637<br />
United States<br />
dea@uchicago.edu<br />
Rieko ASAI<br />
Department <strong>of</strong> Physiological Chemistry<br />
and Metabolism<br />
The <strong>University</strong> <strong>of</strong> Tokyo<br />
7-3-1<br />
Hongo, Bunkyo-ku<br />
Tokyo, Japan<br />
shallow@m.u-tokyo.ac.jp<br />
180<br />
Sophie Astr<strong>of</strong><br />
Department <strong>of</strong> Medicine<br />
Thomas Jefferson <strong>University</strong><br />
1025 Walnut street<br />
Room 312<br />
Philadelphia, PA, 19107<br />
United States<br />
sophie.astr<strong>of</strong>@gmail.com<br />
Mohamad Azhar<br />
Department <strong>of</strong> Pediatrics<br />
Indiana <strong>University</strong> Purdue <strong>University</strong><br />
Indianapolis<br />
Indianapolis, IN, 46202<br />
United States<br />
mazhar@iupui.edu<br />
David Bader<br />
Department <strong>of</strong> Cardiovascular Medicine<br />
Vanderbilt <strong>University</strong><br />
348 Preston Research Building<br />
2220 Pierce Avenue<br />
Nashville, Tennessee, 37232-6300<br />
United States<br />
david.bader@vanderbilt.edu<br />
Yan Bai<br />
Department <strong>of</strong> Molecular Physiology<br />
and Biophysics<br />
Baylor College <strong>of</strong> Medicine<br />
One Baylor Plaza<br />
Anderson Building R504E<br />
Housont, Texas, 77030<br />
United States<br />
yanb@bcm.edu<br />
Jeroen Bakkers<br />
Department <strong>of</strong> Cardiac Development<br />
and Genetics<br />
Hubrecht Institute and <strong>University</strong><br />
Medical Center Utrecht<br />
Uppsalalaan, 8<br />
Utrecht, 3584 CT<br />
Netherlands<br />
j.bakkers@hubrecht.eu<br />
Antonio Baldini<br />
Institute <strong>of</strong> Genetics and Biophysics,<br />
CNR<br />
Via Pietro Castellino 111<br />
Napoli, 80131<br />
Italy<br />
baldini@igb.cnr.it<br />
Toshihiro Banjo<br />
Department <strong>of</strong> Department <strong>of</strong><br />
Developmental Neurobiology<br />
Institute <strong>of</strong> Development, Aging and<br />
Cancer (IDAC), Tohoku university<br />
4-1, Seiryo, Aoba<br />
Sendai, Miyagi, 980-8575<br />
Japan<br />
t-banjo@idac.tohoku.ac.jp
Phil Barnett<br />
Department <strong>of</strong> Anatomy, Emrbyology<br />
and Physiology<br />
HFRC-AMC<br />
Academic Medical Center<br />
Meibergdreef 9<br />
Amsterdam, Netherlands<br />
1105 AZ<br />
p.barnett@amc.uva.nl<br />
Damien Barnette<br />
Department <strong>of</strong> Molecular and Cellular<br />
Pharmacology<br />
<strong>University</strong> <strong>of</strong> Miami Miller School <strong>of</strong><br />
Medicine<br />
1600 NW 10th Ave<br />
Miami, FL, 33136<br />
United States<br />
DBarnette@med.miami.edu<br />
Heather Bartlett<br />
Department <strong>of</strong> Pediatric Cardiology<br />
<strong>University</strong> <strong>of</strong> Iowa Children's Hospital<br />
200 Hawkins Drive<br />
2801 JPP<br />
Iowa City, Iowa, 52242<br />
United States<br />
heather-bartlett@uiowa.edu<br />
Margaret Benny Klimek<br />
Department <strong>of</strong> Molecular, Cellular and<br />
Developmental Biology<br />
<strong>University</strong> <strong>of</strong> Miami, Miller School <strong>of</strong><br />
Medicine<br />
Rosenstiel Medical Sciences Building,<br />
Room 4026/4027<br />
1600 NW 10th Avenue<br />
Miami, FL, 33136<br />
United States<br />
mbenny@med.miami.edu<br />
Subarna Bhattacharya<br />
Biochemistry and Biotechnology and<br />
Bioengineering Center<br />
Medical College <strong>of</strong> Wisconsin<br />
Department <strong>of</strong> Biochemistry, BSB 335<br />
8701 W. Watertown Plank Road<br />
Milwaukee, WI, 53226<br />
United States<br />
bhattacharya@mcw.edu<br />
Brent Bisgrove<br />
Department <strong>of</strong> Neurobiology and<br />
Anatomy<br />
<strong>University</strong> <strong>of</strong> Utah<br />
Eccles Institute <strong>of</strong> Human Genetics<br />
15 North 2030 East, Room 3160<br />
Salt Lake City, Utah, 84106<br />
United States<br />
bbisgrove@genetics.utah.edu<br />
Brian Black<br />
Cardiovascular Research Institute<br />
UCSF<br />
555 Mission Bay Blvd South, MC 3120<br />
San Francisco, CA, 94158-2517<br />
United States<br />
brian.black@ucsf.edu<br />
Marko Boskovski<br />
School <strong>of</strong> Medicine<br />
Yale <strong>University</strong><br />
333 Cedar St. FMP 425<br />
New Haven, CT, 06510<br />
United States<br />
marko.boskovski@yale.edu<br />
Kevin Bosse<br />
Center for Cardiovascular and<br />
Pulmonary Research<br />
The Research Institute at Nationwide<br />
Children's Hospital<br />
700 Children's Drive<br />
Columbus, OH, 43205<br />
United States<br />
kevin.bosse@nationwidechildrens.org<br />
Caitlin Braitsch<br />
Department <strong>of</strong> Molecular<br />
Cardiovascular Biology<br />
Cincinnati Children's Hospital Medical<br />
Center<br />
240 Albert Sabin Way ML 7020<br />
Cincinnati, OH, 45229<br />
United States<br />
caitlin.maynard@cchmc.org<br />
Thomas Brand<br />
National Heart And Lung Institute<br />
Imperial College London<br />
Harefield Heart Science Centre<br />
Hill End Road<br />
Harefield, Middlesex, UB9 6JH<br />
United Kingdom<br />
t.brand@imperial.ac.uk<br />
Michael Bressan<br />
Cardiovascular Research Institute<br />
<strong>University</strong> <strong>of</strong> California San Francisco<br />
555 mission bay blvd south, rm312<br />
San francisco , CA, 94158<br />
United States<br />
Michael.bressan@ucsf.edu<br />
Laura Briggs<br />
Department <strong>of</strong> Regen Medicine and<br />
Cell Bio<br />
MUSC<br />
173 Ashley Ave<br />
BSB601<br />
Charleston, sc, 29425<br />
United States<br />
leb41@musc.edu<br />
Matt Brody<br />
Department <strong>of</strong> Cell and Regenerative<br />
Biology<br />
<strong>University</strong> <strong>of</strong> Wisconsin-Madison<br />
1300 <strong>University</strong> Ave.<br />
322 SMI<br />
Madison, WI, 53706<br />
United States<br />
mjbrody@wisc.edu<br />
181<br />
Derrick Broka<br />
Department <strong>of</strong><br />
Pharmacology/Toxicology<br />
<strong>University</strong> <strong>of</strong> Arizona<br />
1703 E Mabel<br />
Skaggs rm 243<br />
Tucson, AZ, 85719<br />
United States<br />
broka@pharmacy.arizona.edu<br />
Michael Broman<br />
Department <strong>of</strong> Internal Medicine<br />
<strong>University</strong> <strong>of</strong> <strong>Chicago</strong><br />
5841 S Maryland Ave. MC6088<br />
G-608<br />
<strong>Chicago</strong>, IL, 60637<br />
United States<br />
michael.broman@uchospitals.edu<br />
Chris Brown<br />
Department <strong>of</strong> Pediatrics and<br />
Pharmacology<br />
Vanderbilt <strong>University</strong> Medical Center<br />
2213 Garland Ave.<br />
9435-A MRB4 Langford<br />
Nashville, TN, 37232-0493<br />
United States<br />
chris.brown@vanderbilt.edu<br />
Nigel Brown<br />
Department <strong>of</strong> Biomedical Sciences<br />
St George's, <strong>University</strong> <strong>of</strong> London<br />
Cranmer Terrace<br />
Tooting, London, SW17 0RE<br />
United Kingdom<br />
nbrown@sgul.ac.uk<br />
Martina Brueckner<br />
Department <strong>of</strong> Pediatrics<br />
Yale <strong>University</strong><br />
333 Cedar St. FMP 425<br />
New Haven, CT, 06511<br />
United States<br />
martina.brueckner@yale.edu<br />
Benoit Bruneau<br />
Department <strong>of</strong> Cardiovascular Disease<br />
Gladstone Institute<br />
1650 Owens Street<br />
4th floor<br />
San Francisco, CA, 94127<br />
United States<br />
bbruneau@gladstone.ucsf.edu<br />
Luca Brunelli<br />
Department <strong>of</strong> Pediatrics, Division <strong>of</strong><br />
Neonatology<br />
<strong>University</strong> <strong>of</strong> Utah<br />
Williams Building<br />
295 Chipeta Way, Room 2N100<br />
Salt Lake City, UT, 84108<br />
United States<br />
luca.brunelli@hsc.utah.edu
Rachel Brunner<br />
Department <strong>of</strong> Biological Sciences<br />
<strong>University</strong> <strong>of</strong> Illinois at <strong>Chicago</strong><br />
900 S Ashland Ave<br />
Rm4168<br />
<strong>Chicago</strong>, IL, 60607<br />
United States<br />
rbrunn2@uic.edu<br />
Jan Buikema<br />
Cardiovascular Research Institute<br />
Massachusetts General Hospital<br />
185 Cambridge St<br />
Boston, Massachusetts, 02114<br />
United States<br />
buikema.jan@mgh.harvard.edu<br />
Ozanna Burnicka-Turek<br />
Departments <strong>of</strong> Pediatrics, Section <strong>of</strong><br />
Cardiology<br />
The <strong>University</strong> <strong>of</strong> <strong>Chicago</strong>,<br />
900 East 57th Street, KCBD Room<br />
5240<br />
<strong>Chicago</strong>, Illinois, IL-60637<br />
United States<br />
burnickatureko@uchicago.edu<br />
Caroline Burns<br />
Cardiovascular Research Center<br />
(CVRC)<br />
Massachusetts General Hospital<br />
149 13th Street<br />
4th Floor, CVRC, 4138E<br />
Charlestown, MA, 02129<br />
United States<br />
cburns6@partners.org<br />
Jonathan Butcher<br />
Department <strong>of</strong> Biomedical Engineering<br />
Cornell <strong>University</strong><br />
304 Weill Hall<br />
Ithaca, NY, 14850<br />
United States<br />
jtb47@cornell.edu<br />
Todd Camenisch<br />
Department <strong>of</strong> pharmacology and<br />
toxicology<br />
<strong>University</strong> <strong>of</strong> Arizona<br />
College <strong>of</strong> Pharmacy<br />
1703 E. Mabel st. room 234<br />
Tucson, AZ, 85721<br />
camenisch@pharmacy.arizona.edu<br />
Michel Cameron<br />
Department <strong>of</strong> Pharmacology<br />
<strong>University</strong> <strong>of</strong> Montreal, CHU Sainte-<br />
Justine<br />
3175 chemin Cote-Sainte-Catherine<br />
Room 2726<br />
Montreal, Quebec, H3T1C5<br />
Canada<br />
michel.cameron@gmail.com<br />
Marina Campione<br />
Institute <strong>of</strong> Neurosciences<br />
CNR<br />
Viale G. Colombo, 3<br />
Padova, , 35121<br />
Italy<br />
campione@bio.unipd.it<br />
Yunshan Cao<br />
Model Animal Research Center<br />
Nanjing <strong>University</strong><br />
12 Xuefu Road, Pukou<br />
Nanjing, Jiangsu, 210061<br />
China<br />
yunshancao@126.com<br />
Paola Cattaneo<br />
Dip.Scienze Chirurgiche<br />
Universita' Milano-Bicocca<br />
Via Cadore 48<br />
Monza, Mb, 20052<br />
Italy<br />
paolal.cattaneo@gmail.com<br />
Susana Cavallero<br />
Broad Center for Regenerative<br />
Medicine and Stem Cell Research<br />
<strong>University</strong> <strong>of</strong> Southern California<br />
1425 San Pablo St Room 505<br />
Los Angeles, CA, 90033<br />
United States<br />
ccavalle@usc.edu<br />
Barbara Celona<br />
CVRI<br />
UCSF<br />
555 Mission Bay Blvd South<br />
room 382<br />
San Francisco, CA, 94158<br />
United States<br />
barbara.celona@ucsf.edu<br />
Chiann-mun Chen<br />
Department <strong>of</strong> Cardiovascular Medicine<br />
<strong>University</strong> <strong>of</strong> Oxford<br />
Wellcome Trust Centre for Human<br />
Genetics<br />
Roosevelt Drive<br />
Oxford, Oxfordshire, OX3 7BN<br />
United Kingdom<br />
chiann-mun.chen@cardiov.ox.ac.uk<br />
Dongying Chen<br />
Department <strong>of</strong> Pathology, Anatomy and<br />
Cell Biology<br />
Thomas Jefferson <strong>University</strong><br />
1025 Walnut St<br />
Center for Translational Medicine,<br />
Room 301<br />
Philadelphia, PA, 19107<br />
United States<br />
Dongying.Chen@jefferson.edu<br />
182<br />
Jau-Nian Chen<br />
Department <strong>of</strong> Molecular, Cell and<br />
Developmental Biology<br />
UCLA<br />
615 Charles E. Young Drive, South<br />
Los Angeles, California, 90095<br />
United States<br />
chenjn@mcdb.ucla.edu<br />
Li Chen<br />
Department <strong>of</strong> Stem Cell Engineering<br />
Texas Heart Institute<br />
6770 Bertner Ave.<br />
Houston, TX, 77030<br />
United States<br />
lichen@texasheart.org<br />
Hanying Chen<br />
Department <strong>of</strong> Pediatrics<br />
Indiana <strong>University</strong> Purdue <strong>University</strong><br />
Indianapolis<br />
1044 W. Walnut Street<br />
R4 - 312<br />
Indianapolis, IN, 46202<br />
Uni<br />
hanchen@iupui.edu<br />
Neil Chi<br />
Department <strong>of</strong> Medicine<br />
UCSD<br />
9500 Gilman Dr.<br />
Mail code 0613J<br />
La Jolla, CA, 92093<br />
United States<br />
nchi@ucsd.edu<br />
Alvin Chin<br />
Department <strong>of</strong> Pediatrics<br />
Univ <strong>of</strong> Pennsylvania School <strong>of</strong><br />
Medicine/Children's Hospital <strong>of</strong><br />
Philadelphia<br />
Division <strong>of</strong> Cardiology<br />
34th Street and Civic Center Boulevard<br />
Philadelphia, PA, 19096<br />
United States<br />
chinalvi@mail.med.upenn.edu<br />
Sophie Colombo<br />
Department <strong>of</strong> Pediatric Cardiology<br />
Columbia <strong>University</strong> Medical Center<br />
630 W 168th Street<br />
P&S 9-401<br />
New York, New York, 10032<br />
United States<br />
sc3330@columbia.edu<br />
Simon Conway<br />
Program in Developmental Biology &<br />
Neonatal Medicine<br />
Indiana <strong>University</strong>-Purdue <strong>University</strong><br />
Indianapolis<br />
1044 W Walnut Street<br />
siconway@iupui.edu
Jason Cowan<br />
Department <strong>of</strong> Molecular and<br />
Cardiovascular Biology<br />
<strong>University</strong> <strong>of</strong> Cincinnati / CCHMC<br />
240 Albert Sabin Way<br />
MLC 7020<br />
Cincinnati, OH, 45229<br />
United States<br />
Jason.Cowan@cchmc.org<br />
Enrico D'Aniello<br />
Department <strong>of</strong> Molecular<br />
Cardiovascular Biology<br />
Cincinnati Children's Hospital Medical<br />
Center<br />
3333 Burnet Avenue<br />
Cincinnati, Ohio, 45229<br />
United States<br />
enrico.daniello@cchmc.org<br />
José Luis de la Pompa<br />
Cardiovascular and Development<br />
Department<br />
Fundacion CNIC<br />
Calle Melchor Fernandez Almagro, 3<br />
Madrid, Madrid, 28029<br />
Spain<br />
jlpompa@cnic.es<br />
Annemarieke De Vlaming<br />
Department <strong>of</strong> Regenerative Medicine<br />
And Cell Biology<br />
Medical <strong>University</strong> Of South Carolina<br />
171 Ashley Avenue<br />
Bsb-601<br />
Charleston, Sc, 29425<br />
United States<br />
Ellen Dees<br />
Department <strong>of</strong> Pediatrics, Cardiology<br />
Vanderbilt<br />
2200 Children's Way<br />
Suite 5230<br />
Nashville, TN, 37232<br />
United States<br />
ellen.dees@vanderbilt.edu<br />
Karl Degenhardt<br />
Department <strong>of</strong> pediatrics<br />
Children's Hospital <strong>of</strong> Philadelphia<br />
34th and Civic Center Blvd<br />
Philadelphia, PA, 19103<br />
United States<br />
degenhardt@email.chop.edu<br />
Daniel DeLaugher<br />
Department <strong>of</strong> Cell & Developmental<br />
Biology<br />
Vanderbilt <strong>University</strong> Medical Center<br />
460 Preston Research Building<br />
Nashville, TN, 37232-6600<br />
United States<br />
daniel.m.delaughter@vanderbilt.edu<br />
Ashish Deshwar<br />
Department <strong>of</strong> Molecular Genetics<br />
<strong>University</strong> <strong>of</strong> Toronto<br />
Rm 11-601B, Toronto Medical<br />
Discovery Tower<br />
101 College St.<br />
Toronto, ON, M5G 1L7<br />
Canada<br />
ashish.deshwar@utoronto.ca<br />
Patrick Devine<br />
Gladstone Institute <strong>of</strong> Cardiovascular<br />
Disease<br />
J. David Gladstone Institute<br />
1650 Owens Street<br />
San Francisco, CA, 94158<br />
United States<br />
patrick.devine@gladstone.ucsf.edu<br />
Tracy Dohn<br />
Department <strong>of</strong> Molecular and<br />
Cardiovascular Biology<br />
Cincinnati Children's Hospital<br />
3333 Burnet Ave.<br />
Cincinnati, OH, 45229<br />
United States<br />
tracy.dohn@cchmc.org<br />
Jorge N Dominguez Macias<br />
Department Of Experimental Biology<br />
<strong>University</strong> Of Jaén<br />
Faculty Of Experimental Sciences,<br />
Building B3<br />
Paraje De Las Lagunillas S/N<br />
Jaen, , 23071<br />
Spain<br />
jorgendm@ujaen.es<br />
Kerry Dorr<br />
Department <strong>of</strong> Genetics<br />
<strong>University</strong> <strong>of</strong> North Carolina<br />
221 Fordham Hall<br />
Medical Drive, CB#3280<br />
Chapel Hill, NC, 27599<br />
United States<br />
kdehghan@email.unc.edu<br />
Laurent Dupays<br />
Department <strong>of</strong> Developmental Biology<br />
National Institute for Medical Research<br />
The Ridgeway, Mill Hill<br />
London, , NW7 1AA<br />
United Kingdom<br />
ldupays@nimr.mrc.ac.uk<br />
Laura Dyer<br />
Department <strong>of</strong> McAllister Heart Institute<br />
<strong>University</strong> <strong>of</strong> North Carolina at Chapel<br />
Hill<br />
2320 MBRB CB#7126<br />
111 Mason Farm Road<br />
Chapel Hill, NC, 27599<br />
United States<br />
ldyer1@email.unc.edu<br />
183<br />
Sylvia Evans<br />
Skaggs School <strong>of</strong> Pharmacy and<br />
Department <strong>of</strong> Medicine<br />
<strong>University</strong> <strong>of</strong> California San Diego<br />
9500 Gilman Drive<br />
Biomedical Sciences Building Rm 5024<br />
Mail Code 0613-C<br />
La Jolla CA, CA, 92093<br />
United States<br />
syevans@ucsd.edu<br />
Heather Evans-Anderson<br />
Department <strong>of</strong> Biology<br />
Winthrop <strong>University</strong><br />
102 Dalton Hall<br />
Rock Hill, SC, 29733<br />
United States<br />
evansandh@winthrop.edu<br />
Ming Fang<br />
Molecular Cardiovascular Biology/Heart<br />
Institute<br />
Cincinnati Children's Hospital Research<br />
Fundation<br />
240 Albert Sabin Way MLC 7020<br />
Cincinnati, OH, 45229<br />
United States<br />
ming.fang@cchmc.org<br />
Yi Fang<br />
Department <strong>of</strong> Cell Biology<br />
Duke <strong>University</strong> Medical Center and<br />
Howard Hughes Medical Institutes<br />
353 Nanaline Duke Building<br />
Box 3709<br />
Durham, NC, 27710<br />
United States<br />
yi.fang@dm.duke.edu<br />
Deborah Farkas<br />
Department <strong>of</strong> Developmental Biology<br />
<strong>University</strong> <strong>of</strong> Pittsburgh School <strong>of</strong><br />
Medicine<br />
530 45th Street<br />
8162 Rangos Research Building<br />
Pittsburgh, PA, 15201<br />
United States<br />
hamburg@pitt.edu<br />
Qingping Feng<br />
Department <strong>of</strong> Physiology and<br />
Pharmacology<br />
<strong>University</strong> <strong>of</strong> Western Ontario<br />
Medical Sciences Building<br />
Room M254<br />
London, Ontario, N6A 5C1<br />
Canada<br />
qfeng@uwo.ca<br />
Jason Fish<br />
Department <strong>of</strong> Cell & Molecular Biology<br />
<strong>University</strong> Health Network, Toronto<br />
General Research Institute<br />
101 College Street, MaRS Centre,<br />
TMDT 3-308<br />
Toronto, Ontario, M5G 1L7<br />
Canada<br />
jason.fish@utoronto.ca
Steven Fisher<br />
Department <strong>of</strong> medicine<br />
<strong>University</strong> <strong>of</strong> Maryland<br />
S-012c HSF II<br />
20 S Penn St<br />
baltimore, md, 21201<br />
United States<br />
SFisher1@medicine.umaryland.edu<br />
Richard Francis<br />
Department <strong>of</strong> Developmental Biology<br />
<strong>University</strong> <strong>of</strong> Pittsburgh School <strong>of</strong><br />
Medicine<br />
530 45th Street<br />
8119 Rangos Research Center<br />
Pittsburgh, PA, 15201<br />
United States<br />
rfrancis@pitt.edu<br />
Diego Franco<br />
Department <strong>of</strong> Experimental Biology<br />
<strong>University</strong> <strong>of</strong> Jaen<br />
CU Las Lagunillas s/n<br />
jaen, jaen, 23071<br />
Spain<br />
dfranco@ujaen.es<br />
Vidu Garg<br />
Department <strong>of</strong> Pediatrics/Center for<br />
Cardiovascular and Pulmonary<br />
Research<br />
Ohio State <strong>University</strong>/Nationwide<br />
Children's Hospital<br />
700 Children's Drive W302<br />
Columbus, OH, 43205<br />
United States<br />
vidu.garg@nationwidechildrens.org<br />
Joseph Gawdzik<br />
Department <strong>of</strong> Pharmaceutical Science<br />
<strong>University</strong> <strong>of</strong> Wisconsin<br />
777 Highland Avenue<br />
Madison, WI, 53705<br />
United States<br />
gawdzik@wisc.edu<br />
Stefan Geyer<br />
Department <strong>of</strong> Center for Anatomy and<br />
Cell Biology<br />
Medical <strong>University</strong> <strong>of</strong> Vienna<br />
Waehringer Str. 13<br />
Vienna, , 1090<br />
Austria<br />
stefan.geyer@meduniwien.ac.at<br />
Shibnath Ghatak<br />
Department <strong>of</strong> Regenerative Medicine<br />
& Cell Biology<br />
Medical <strong>University</strong> <strong>of</strong> South Carolina<br />
BSB-601, PO Box 250508<br />
173 Ashley Avenue<br />
Charleston, SC, 29403<br />
United States<br />
ghatak@musc.edu<br />
Nikki Glenn<br />
Department <strong>of</strong> Developmental Biology<br />
Cincinnati Children's Hospital<br />
240 Albert Sabin Way<br />
Cincinnati, OH, 45229<br />
United States<br />
nikki.glenn@cchmc.org<br />
Vicky Gomez<br />
Department <strong>of</strong> Molecular<br />
Cardiovascular Biology<br />
Cincinnati Children's Hospital Research<br />
Foundation<br />
240 Albert Sabin Way MLC 7020<br />
Cincinnati, OH, 45229<br />
United States<br />
maria.gomez@cchmc.org<br />
Bushra Gorsi<br />
Department <strong>of</strong> Neurobiology and<br />
Anatomy<br />
<strong>University</strong> <strong>of</strong> Utah<br />
Eccles Institute <strong>of</strong> Human Genetics<br />
15N 2030E<br />
Salt lake city, Utah, 84102<br />
United States<br />
bgorsi@genetics.utah.edu<br />
Stephanie Greene<br />
Department <strong>of</strong> Molecular Physiology<br />
and Biophysics<br />
Baylor College <strong>of</strong> Medicine<br />
1 Baylor Plaza, 504E<br />
Houston, TX, 77030<br />
United States<br />
sbgreene@bcm.edu<br />
Franziska Greulich<br />
Department <strong>of</strong> Molecular Biology<br />
Hannover Medical School<br />
Carl Neuberg Str. 1<br />
Hannover, , 30625<br />
Germany<br />
greulich.franziska@mh-hannover.de<br />
Paul Grossfeld<br />
Department <strong>of</strong> Pediatric Cardiology<br />
UCSD School <strong>of</strong> Medicine<br />
3020 Children's Way, MC 5004<br />
San Diego, Cal, 92123<br />
United States<br />
pgrossfeld@ucsd.edu<br />
Shi Gu<br />
Department <strong>of</strong> Biomedical Engineering<br />
Case Western Reserve <strong>University</strong><br />
11100 Euclid Ave.<br />
Wearn Room# 344<br />
Cleveland, OH, 44106<br />
United States<br />
sxg181@case.edu<br />
184<br />
Burcu Guner-Ataman<br />
Department <strong>of</strong> Cardiovascular<br />
Research Center<br />
Massachusetts General Hospital,<br />
Harvard Medical School<br />
149 13th Street<br />
CVRC, 4th floor, 4138E<br />
Charlestown, MA, 02129<br />
United States<br />
bguner@cvrc.mgh.harvard.edu<br />
Vikas Gupta<br />
Department <strong>of</strong> Cell Biology<br />
Duke <strong>University</strong> Medical Center<br />
353 Nanaline Duke<br />
Research Drive<br />
Durham, NC, 27710<br />
United States<br />
vg24@duke.edu<br />
Zoltan Hajdu<br />
Department <strong>of</strong> Regenerative Medicine<br />
and Cell Biology<br />
Medical <strong>University</strong> <strong>of</strong> South Carolina<br />
173 Ashley Avenue CRI605<br />
Mailstop 250508<br />
Charleston , SC, 29425<br />
United States<br />
hajdu@musc.edu<br />
Rami Halabi<br />
Department <strong>of</strong> Physiology and<br />
Pharmacology<br />
Western <strong>University</strong> Canada<br />
1151 Richmond Street<br />
London, Ontario, N6A3K7<br />
Canada<br />
rhalabi5@gmail.com<br />
Mingda Han<br />
Department <strong>of</strong> Pediatrics<br />
<strong>University</strong> <strong>of</strong> South Florida<br />
Children's Research Institute<br />
140 7th Avenue South, CRI-2007<br />
Saint Petersburg, FL, 33701<br />
United States<br />
mhan@health.usf.edu<br />
Keerthi Harikrishnan<br />
Department <strong>of</strong> Regenerative and Cell<br />
Biology<br />
Medical <strong>University</strong> <strong>of</strong> South Carolina<br />
173 Ashley Avenue<br />
BSB 653<br />
Charleston, SC, 29425<br />
United States<br />
harikris@musc.edu<br />
Richard Harvey<br />
Department <strong>of</strong> Developmental and<br />
Stem Cell Biology<br />
Victor Chang Cardiac Research<br />
Institute<br />
405 Liverpool Street<br />
Darlinghurst<br />
Sydney, NSW, 2010<br />
Australia<br />
r.harvey@victorchang.edu.au
Aibin He<br />
Department <strong>of</strong> Cardiology<br />
Children's Hospital Boston & Harvard<br />
Medical School<br />
300 Longwood Ave.<br />
Boston, MA, 02115<br />
United States<br />
ahe@enders.tch.harvard.edu<br />
Jonathon Hill<br />
Department <strong>of</strong> Neurobiology and<br />
Anatomy<br />
<strong>University</strong> <strong>of</strong> Utah<br />
15 N 2030 E<br />
EIHG Room 3260<br />
Salt Lake City, UT, 84112<br />
United States<br />
jhill@genetics.utah.edu<br />
Robert Hinton<br />
Heart Institute<br />
Cincinnati Childrens<br />
3333 Burnet Ave<br />
MLC 2003<br />
Cincinnati, Oh, 45229<br />
United States<br />
robert.hinton@cchmc.org<br />
Andrew H<strong>of</strong>fmann<br />
Committee on Development,<br />
Regeneration and Stem Cell Biology<br />
<strong>University</strong> <strong>of</strong> <strong>Chicago</strong><br />
900 E 57th Street<br />
Room 5240 LB-5<br />
<strong>Chicago</strong>, IL, 60657<br />
United States<br />
adh<strong>of</strong>fma@uchicago.edu<br />
Peter H<strong>of</strong>steen<br />
Department <strong>of</strong> Pharmacy<br />
<strong>University</strong> <strong>of</strong> Wisconsin<br />
777 highland ave.<br />
Madison, WI, 53705<br />
United States<br />
ph<strong>of</strong>steen@wisc.edu<br />
Alisha Holloway<br />
Gladstone Institutes<br />
1650 Owens Street<br />
San Francisco, CA, 94158<br />
United States<br />
alisha.holloway@gladstone.ucsf.edu<br />
Brandon Holtrup<br />
Department <strong>of</strong> Pediatics<br />
Children's Memorial Research Center<br />
CMRC<br />
2430 N Halsted St<br />
<strong>Chicago</strong> , Illinois, 60614<br />
United States<br />
brandon.holtrup@gmail.com<br />
Mary Holtz<br />
Department <strong>of</strong> Biochemistry<br />
Medical College <strong>of</strong> Wisconsin<br />
8701 Watertown Plank Road<br />
Milwaukee, Wisconsin, 53226<br />
United States<br />
mlholtz@mcw.edu<br />
Jianxin Hu<br />
Cardiovascular research institute<br />
Mail Code 3120, Smith Cardiovascular<br />
Research Building<br />
555 Mission Bay Blvd. South<br />
San Francisco, California, 94158<br />
United States<br />
jianxin.hu@ucsf.edu<br />
Dirk Hubmacher<br />
Department <strong>of</strong> Biomedical Engineering<br />
Cleveland Clinic, Lerner Research<br />
Institute<br />
9500 Euclid Avenue<br />
ND4-22a<br />
Cleveland, OH, 44195<br />
United States<br />
hubmacd@ccf.org<br />
Danielle Huk<br />
Center for Cardiovascular and<br />
Pulmonary Research<br />
The Research Institute at Nationwide<br />
Children's Hospital<br />
700 Children's Drive<br />
Columbus, OH, 43205<br />
United States<br />
danielle.huk@nationwidechildrens.org<br />
Mary Hutson<br />
Department <strong>of</strong> Pediatrics<br />
Duke <strong>University</strong><br />
Box 103105, DUMC<br />
Jones Bldg<br />
Durham, NC, 27710<br />
United States<br />
mhutson@duke.edu<br />
Akira Imamoto<br />
Ben May Department for Cancer<br />
Research<br />
The <strong>University</strong> <strong>of</strong> <strong>Chicago</strong><br />
929 E 57th Street, GCIS-W332<br />
<strong>Chicago</strong>, IL, 60637<br />
United States<br />
aimamoto@uchicago.edu<br />
Kyoko Imanaka-Yoshida<br />
Department <strong>of</strong> Pathology and Matrix<br />
Biology<br />
Mie <strong>University</strong> Graduate School <strong>of</strong><br />
Medicine<br />
2-174 Edobashi<br />
Tsu, Mie, 514-8507<br />
Japan<br />
imanaka@doc.medic.mie-u.ac.jp<br />
185<br />
Jose Frnacisco Islas<br />
Department <strong>of</strong> Stem Cell Eng<br />
Texas Heart Inst<br />
6770 Bertner<br />
MC2-225<br />
Houston, Texas, 77030<br />
United States<br />
jislas@texasheart.org<br />
Patrick Jay<br />
Department <strong>of</strong> Pediatrics<br />
Washington <strong>University</strong> School <strong>of</strong><br />
Medicine<br />
Box 8208<br />
660 South Euclid Avenue<br />
St. Louis, MO, 63110<br />
United States<br />
jay_p@kids.wustl.edu<br />
Audrey Jerde<br />
Committee on Development,<br />
Regeneration, and Stem Cell Biology<br />
<strong>University</strong> <strong>of</strong> <strong>Chicago</strong><br />
5841 S. Maryland Ave<br />
AMB G-608 MC 6088<br />
<strong>Chicago</strong>, Illinois, 60637<br />
United States<br />
ajerde@uchicago.edu<br />
Guangju Ji<br />
Institute <strong>of</strong> Biophysics<br />
chinese Academy <strong>of</strong> Sciences<br />
15 datun road<br />
Beijing China<br />
100101<br />
gj28@ibp.ac.cn<br />
Yongchang Ji<br />
Department <strong>of</strong> Cell and developmental<br />
biology<br />
SUNY upstate medical university<br />
750 East Adams Street<br />
syracuse, NY, 13210<br />
United States<br />
jiy@upstate.edu<br />
Kai Jiao<br />
Department <strong>of</strong> Genetics<br />
The Univ. <strong>of</strong> Alabama at Birmingham<br />
720 20th St. S.<br />
768 Kaul Building<br />
Birmingham, AL, 35294<br />
United States<br />
kjiao@uab.edu<br />
Ran Jing<br />
Department <strong>of</strong> medicine<br />
university <strong>of</strong> california, san diego<br />
9500 Gilman drive BSB 5025<br />
La jolla, CA<br />
United States<br />
rjing@ucsd.edu
Chuanchau Jou<br />
Department <strong>of</strong> Pediatric Cardiology<br />
<strong>University</strong> <strong>of</strong> Utah<br />
Primary Children's Medical Center<br />
100 N. Mario Capecchi Drive, Suite<br />
1500<br />
Salt Lake City, Utah, 84113<br />
United States<br />
jerry_jou@hotmail.com<br />
Jerry Jou<br />
Department <strong>of</strong> Pediatric Cardiology<br />
<strong>University</strong> <strong>of</strong> Utah<br />
Primary Children's Medical Center<br />
100 N. Mario Capecchi Drive, Suite<br />
1500<br />
Salt Lake City, Utah, 84113<br />
United States<br />
jerry_jou@hotmail.com<br />
Vesa Kaartinen<br />
Department <strong>of</strong> Biologic & Materials<br />
Sciences<br />
<strong>University</strong> <strong>of</strong> Michigan<br />
1011 N. <strong>University</strong><br />
DENT 2305<br />
Ann Arbor, MI, 48109<br />
United States<br />
vesak@umich.edu<br />
Ganga Karunamuni<br />
Department <strong>of</strong> Pediatrics<br />
Case Western Reserve <strong>University</strong><br />
10900 Euclid Avenue<br />
Cleveland, OH, 44106<br />
United States<br />
ghk6@case.edu<br />
Bogac Kaynak<br />
Developmental Biology Program<br />
Insitute <strong>of</strong> Biotechnology, <strong>University</strong> <strong>of</strong><br />
Helsinki<br />
P.O.Box 56 <strong>University</strong> <strong>of</strong> Helsinki<br />
Helsinki, , 00014<br />
Finland<br />
bogac.kaynak@helsinki.fi<br />
Robert Kelly<br />
Department <strong>of</strong> Developmental Biology<br />
Institute <strong>of</strong> Marseille Luminy<br />
Aix-Marseille <strong>University</strong><br />
Campus de Luminy Case 907<br />
Marseille, , 13288<br />
France<br />
Robert.Kelly@univ-amu.fr<br />
Doreswamy Kenchegowda<br />
Department <strong>of</strong> Medicine (Cardiology)<br />
<strong>University</strong> <strong>of</strong> Maryland School <strong>of</strong><br />
Medicine<br />
20 Penn Street<br />
HSF II, S007<br />
Baltimore, MD, 21201<br />
United States<br />
k.doreswamy@gmail.com<br />
Christi Kern<br />
Department <strong>of</strong> Regenerative Medicine<br />
and Cell Biology<br />
Medical <strong>University</strong> <strong>of</strong> South Carolina<br />
171 Ashley Avenue<br />
Charleston, SC, 29425<br />
United States<br />
kernc@musc.edu<br />
Anna Keyte<br />
Department <strong>of</strong> Pediatrics<br />
Duke <strong>University</strong><br />
402 Jones Building<br />
DUMC Box 103105<br />
Durham, NC, 27710<br />
United States<br />
alk2@duke.edu<br />
Hinako Kidokoro<br />
Department <strong>of</strong> Neurobiology and<br />
Anatomy<br />
<strong>University</strong> <strong>of</strong> Utah, School <strong>of</strong> Medicine<br />
20 North 1900 East, 401 MREB<br />
Salt Lake City, Utah, 84132-3401<br />
United States<br />
hinako.kidokoro@utah.edu<br />
Andrew Kim<br />
Department <strong>of</strong> Developmental Biology<br />
<strong>University</strong> <strong>of</strong> Pittsburgh School <strong>of</strong><br />
Medicine<br />
530 45th St.<br />
8162 Rangos Research Center<br />
Pittsburgh, PA, 15201<br />
United States<br />
akim@pitt.edu<br />
Chul Kim<br />
Department <strong>of</strong> Pediatrics/Section <strong>of</strong><br />
Cardiology<br />
<strong>University</strong> <strong>of</strong> <strong>Chicago</strong><br />
900 E 57th street<br />
5240-LB1<br />
<strong>Chicago</strong>, IL, 60637<br />
United States<br />
chulsdream@uchicago.edu<br />
Eun Young Kim<br />
Center for stem cell engineering<br />
Texas Heart Institute<br />
THI, PO box 20345, MC2-255<br />
Houston, TX, 77030<br />
United States<br />
eun.young.kim.2@uth.tmc.edu<br />
Gene Kim<br />
Department <strong>of</strong> Medicine<br />
<strong>University</strong> <strong>of</strong> <strong>Chicago</strong><br />
5841 S. Maryland Ave<br />
MC 6080<br />
<strong>Chicago</strong>, IL, 60637<br />
United States<br />
gkim1@medicine.bsd.uchicago.edu<br />
186<br />
Jieun Kim<br />
Department <strong>of</strong> Cardiothoracic Surgery<br />
Children's Hospital Los Angeles<br />
4661 Sunset Blvd.<br />
Los Angeles, CA, 90027<br />
United States<br />
jiekim@chla.usc.edu<br />
Ki-Sung Kim<br />
Departmet <strong>of</strong> Physiological Chemistry<br />
and Metabolism<br />
Graduate School <strong>of</strong> Medicine, The<br />
<strong>University</strong> <strong>of</strong> Tokyo<br />
7-3-1 Hongo,<br />
Bunkyo-ku,<br />
Tokyo, , 113-0033<br />
Japan<br />
kkin-tky@umin.ac.jp<br />
Isabelle King<br />
Department <strong>of</strong> Pediatrics<br />
<strong>University</strong> <strong>of</strong> California, San Francisco<br />
505 Parnassus<br />
San Francisco, CA, 94143<br />
United States<br />
iking@gladstone.ucsf.edu<br />
Caroline Kinnear<br />
Department <strong>of</strong> Genetics and Genome<br />
Biology<br />
Hospital for Sick Children<br />
555 <strong>University</strong> Av<br />
7028 McMaster, mital lab<br />
Toronto, Ontario, M5G 1X8<br />
Canada<br />
caroline.kinnear@sickkids.ca<br />
Linda Klotz<br />
Molecular Medicine Unit<br />
<strong>University</strong> College London<br />
30 Guilford Street<br />
UCL Institute <strong>of</strong> Child Health<br />
London, , WC1N 1EH<br />
United Kingdom<br />
l.klotz@ucl.ac.uk<br />
Sara Koenig<br />
Center for Cardiovascular and<br />
Pulmonary Research<br />
Nationwide Children's Hospital<br />
700 Children's Dr.<br />
Columbus, OH, 43205<br />
United States<br />
sara.koenig@nationwidechildrens.org<br />
Kurt Kolander<br />
Department <strong>of</strong> Biochemistry<br />
Medical College <strong>of</strong> Wisconsin<br />
8701 Watertown Plk Rd<br />
Milwaukee, WI, 53226<br />
United States<br />
kkolande@mcw.edu
Kazuko Koshiba-Takeuchi<br />
Division <strong>of</strong> Cardiovascular<br />
Regeneration<br />
Institute <strong>of</strong> Molecular and Cellular<br />
Biosciences<br />
1-1-1 Yayoi<br />
Bunkyo-ku, Tokyo, 113-0032<br />
Japan<br />
koshibatk@iam.u-tokyo.ac.jp<br />
Maike Krenz<br />
Department <strong>of</strong> Medical Pharmacology<br />
and Physiology<br />
<strong>University</strong> <strong>of</strong> Missouri-Columbia<br />
Dalton Cardiovascular Research Center<br />
134 Research Park Dr<br />
Columbia, MO, 65211<br />
United States<br />
krenzm@missouri.edu<br />
Tsutomu Kume<br />
Department <strong>of</strong> Medicine<br />
Northwestern <strong>University</strong> School <strong>of</strong><br />
Medicine<br />
t-kume@northwestern.edu<br />
Hiroki Kurihara<br />
Department <strong>of</strong> Physiologicalchemistry<br />
and Metabolism<br />
The <strong>University</strong> <strong>of</strong> Tokyo<br />
7-3-1<br />
Hongo, Bunkyo-ku<br />
Tokyo, , 113-0033<br />
Japan<br />
shallow1well@msn.com<br />
Junghun Kweon<br />
Department <strong>of</strong> Pediatrics<br />
900, E 57th St, RM 5240<br />
<strong>Chicago</strong>, IL, 60637<br />
United States<br />
jkweon@uchicago.edu<br />
Jacquelyn Lajiness<br />
Department <strong>of</strong> Pediatrics<br />
Indiana <strong>University</strong> Purdue <strong>University</strong><br />
Indianapolis<br />
1044 W. Walnut Street<br />
R4 402F<br />
Indianapolis, IN, 46202<br />
United States<br />
lewisjad@iupui.edu<br />
Edward Lammer<br />
Department <strong>of</strong> Research<br />
Children's Hospital Oakland Research<br />
Institute<br />
5700 Martin Luther King Jr. Way<br />
Oakland, CA, 94609<br />
United States<br />
elammer@chori.org<br />
Eva Lana-Elola<br />
Department <strong>of</strong> Immune Cell Biology<br />
MRC, National Institute for Medical<br />
Research<br />
The Ridgeway<br />
Mill Hill<br />
London, London, NW7 1AA<br />
United Kingdom<br />
eelola@niimr.rmc.ac.uk<br />
Julie Lander<br />
Department <strong>of</strong> Molecular<br />
Cardiovascular Biology<br />
Cincinnati Children's Hospital Medical<br />
Center<br />
240 Albert Sabin Way<br />
MCL7020<br />
Cincinnati, OH, 45229<br />
United States<br />
julie.lander@cchmc.org<br />
Jessica Lauriol<br />
Department <strong>of</strong> Cardiology<br />
Beth Israel Deaconess Medical Center-<br />
Harvard Medical School<br />
CLS Building 917E<br />
3 blackfan circle<br />
Boston, MA, 02115<br />
United States<br />
Jlauriol@bidmc.harvard.edu<br />
Kyu-Ho Lee<br />
Department <strong>of</strong> Pediatric Cardiology<br />
Children's Hospital, Medical <strong>University</strong><br />
<strong>of</strong> South Carolina<br />
MSC-508<br />
173 Ashley Ave<br />
Charleston, SC, 29425<br />
United States<br />
leekh@musc.edu<br />
Mary Lee<br />
Department <strong>of</strong> Molecular<br />
Cardiovascular Biology<br />
Cincinnati Children's Hospital Research<br />
Foundation<br />
240 Albert Sabin Way<br />
MLC 7020<br />
Cincinnati, OH, 45209<br />
United States<br />
mary.lee@cchmc.org<br />
Youngsook Lee<br />
Department <strong>of</strong> Cell and Regenerative<br />
Biology<br />
<strong>University</strong> <strong>of</strong> Wisconsin-Madison<br />
1300 <strong>University</strong> Avenue<br />
Madison, WI, 53706<br />
youngsooklee@wisc.edu<br />
Alejandro Lencinas<br />
Department <strong>of</strong> Cellular Molecular<br />
Medicine<br />
<strong>University</strong> <strong>of</strong> Arizona<br />
1501 North Campbell<br />
Tucson, az, 85724-5044<br />
United States<br />
lencinas@pharmacy.arizona.edu<br />
187<br />
Carmen Leung<br />
Department <strong>of</strong> Physiology &<br />
Pharmacology<br />
<strong>University</strong> <strong>of</strong> Western Ontario<br />
Medical Science Building Rm. 253<br />
London, Ontario, N6A 5C1<br />
Canada<br />
carmz08@gmail.com<br />
Dana Levasseur<br />
Department <strong>of</strong> Internal Medicine<br />
<strong>University</strong> <strong>of</strong> Iowa Carver College <strong>of</strong><br />
Medicine<br />
285 Newton Road<br />
CBRB 3270C<br />
Iowa City, IA, 52242<br />
United States<br />
dana-levasseur@uiowa.edu<br />
Na Li<br />
Yale Cardiovascular Research Center<br />
Yale <strong>University</strong><br />
300 George Street, 7th floor<br />
New Haven, CT, 06511<br />
United States<br />
na.li@yale.edu<br />
Sean Li<br />
Department <strong>of</strong> surgery<br />
Children's Hospital Boston, Harvard<br />
Medical School<br />
300 Longwood Ave<br />
Boston, MA, 02115<br />
United States<br />
sean.li@childrens.harvard.edu<br />
Yanyang Li<br />
Department <strong>of</strong> biological sciences<br />
UIC<br />
900 S Ashland Ave<br />
MBRB building<br />
chicago, IL, 60607<br />
United States<br />
yli98@uic.edu<br />
You Li<br />
Department <strong>of</strong> Developmental Biology<br />
<strong>University</strong> <strong>of</strong> Pittsburgh, School <strong>of</strong><br />
Medicine<br />
8162 Rango's Research Center, 530<br />
45th Street<br />
Pittsburgh, PA, 15201<br />
United States<br />
yol11@pitt.edu<br />
Dong Liang<br />
Center for translational medicine<br />
Thomas Jefferson <strong>University</strong><br />
1025 Walnut Street<br />
Philadelphia, PA, 19107<br />
United States<br />
dong.liang@jefferson.edu
Ching-Ling (Ellen) Lien<br />
Department <strong>of</strong> Surgery<br />
Children's Hospital Los<br />
Angeles/<strong>University</strong> <strong>of</strong> Southern<br />
California<br />
Saban Research Institute<br />
4650 Sunset Blvd. MS#137<br />
Los Angeles, CA, 90027<br />
United States<br />
clien@chla.usc.edu<br />
Fang Lin<br />
Department <strong>of</strong> Anatomy and Cell<br />
Biology<br />
<strong>University</strong> <strong>of</strong> Iowa<br />
51 Newton Rd<br />
Iowa City, IA, 52242<br />
United States<br />
fang-lin@uiowa.edu<br />
Lizhu Lin<br />
Skaggs School <strong>of</strong> Pharmacy<br />
<strong>University</strong> <strong>of</strong> California San Diego<br />
9500 Gilman Dr.<br />
BSB 5027, Mc 0613c<br />
La Jolla, CA, CA 92093<br />
United States<br />
lilin@ucsd.edu<br />
Zhiqiang Lin<br />
Department <strong>of</strong> Cardiology<br />
Children Hospital Boston<br />
300 Longwood Ave, Enders Building<br />
1224<br />
Boston, MA, 02115<br />
United States<br />
zlin@enders.tch.harvard.edu<br />
Joy Lincoln<br />
Center for Cardiovascular and<br />
Pulmonary Research<br />
Nationwide Children's Hospital, The<br />
Ohio State <strong>University</strong><br />
700 Children's Drive<br />
W309<br />
Columbus, OH, 43205<br />
United States<br />
joy.lincoln@nationwidechildrens.org<br />
Stephanie Lindsey<br />
Department <strong>of</strong> Biomedical Engineering<br />
Cornell <strong>University</strong><br />
526 Campus Road<br />
Ithaca, New York, 14850<br />
United States<br />
sel238@cornell.edu<br />
Gary Liu<br />
CVRI<br />
UCSF<br />
555 Mission Bay Blvd South Room 312<br />
San Francisco, CA, 94158<br />
United States<br />
gary.liu@ucsf.edu<br />
Xiaoqin Liu<br />
Department <strong>of</strong> Developmental Biology<br />
<strong>University</strong> <strong>of</strong> Pittsburgh<br />
8112 Rangos Research Center<br />
530 45th Street<br />
Pittsburgh, PA, 15201<br />
United States<br />
liuxq0908@gmail.com<br />
Yun Liu<br />
Department <strong>of</strong> Biology and<br />
Biochemistry<br />
<strong>University</strong> <strong>of</strong> Houston<br />
4800 Calhoun Rd<br />
SR 2, rm 349<br />
Houston, Texas, 77204<br />
United States<br />
yliu54@uh.edu<br />
zhi-ping Liu<br />
Department <strong>of</strong> internal medicine,<br />
cardiology division<br />
UT Southwestern Medical Center<br />
5323 Harry Hines Blvd<br />
Dallas, TX, 75235-8573<br />
United States<br />
zhi-ping.liu@utsouthwestern.edu<br />
Cecilia Lo<br />
Department <strong>of</strong> Developmental Biology<br />
<strong>University</strong> <strong>of</strong> Pittsburgh School <strong>of</strong><br />
Medicine<br />
530 45th St<br />
Pittsburgh, PA, 15201<br />
United States<br />
locgreat@gmail.com<br />
Marie Lockhart<br />
Department <strong>of</strong> Regenerative Medicine<br />
and Cell Biology<br />
Medical <strong>University</strong> <strong>of</strong> South Carolina<br />
173 Ashley Ave<br />
BSB, Suite 601<br />
Charleston, SC, 29407<br />
lockharm@musc.edu<br />
Jonathan Louie<br />
Department <strong>of</strong> Cardiovascular<br />
Research Institute<br />
<strong>University</strong> <strong>of</strong> California San Francisco<br />
555 Mission Bay Blvd. South<br />
Rm. 312<br />
San Francisco, CA, 94158<br />
United States<br />
jonathan.louie@ucsf.edu<br />
Xiangru Lu<br />
Department <strong>of</strong> Physiology and<br />
Pharmacology<br />
<strong>University</strong> <strong>of</strong> Western Ontario<br />
Medical Sciences Building<br />
London, Ontario, N6A 5C1<br />
Canada<br />
sharon.lu@schulich.uwo.ca<br />
188<br />
Guillermo Luxan<br />
Cardiovascular and Developmental<br />
Repair Department<br />
Fundacion CNIC<br />
Calle Melchor Fernandez Almagro, 3<br />
Madrid, Madrid, 28029<br />
Spain<br />
gluxan@cnic.es<br />
Yue Ma<br />
Institute <strong>of</strong> Biophysics; CAS<br />
No. 15 Datun Road<br />
Chaoyang District<br />
Beijing, , 100101<br />
China<br />
yuema@ibp.ac.cn<br />
Uday Bhanu Maachani<br />
Department <strong>of</strong> Cardiology<br />
<strong>University</strong> <strong>of</strong> Maryland<br />
HSF-II room-S007,<br />
20 penn street,<br />
Baltimore, Maryland, 21201<br />
United States<br />
udaybhanumachani@gmail.com<br />
John Mably<br />
Department <strong>of</strong> Cardiology,<br />
Cardiovascular Research<br />
Boston Children's<br />
320 Longwood Avenue<br />
Enders 1276<br />
Boston, MA, 02115<br />
United States<br />
john@mablylab.org<br />
Colin Maguire<br />
Department <strong>of</strong> Neurobiology and<br />
Anatomy<br />
<strong>University</strong> <strong>of</strong> Utah<br />
15 N 2030 East, Rm2280<br />
Salt Lake City, UT, 84112<br />
United States<br />
cmaguire@genetics.utah.edu<br />
Shinji Makino<br />
Department <strong>of</strong> Cardiology<br />
Keio <strong>University</strong> School <strong>of</strong> Medicine<br />
35 Shinanomachi, Shinjuku-ku<br />
Tokyo, , 160-8582<br />
Japan<br />
smakino@z6.keio.jp<br />
Ashok Kumar Manickaraj<br />
Department <strong>of</strong> Genetics and Genome<br />
Biology<br />
Hospital for Sick Children<br />
555 <strong>University</strong> Avenue<br />
Toronto, Ontario, M5G1X8<br />
Canada<br />
ashokkumar.manickaraj@sickkids.ca
Andrea Marion<br />
Department <strong>of</strong> Biological Sciences<br />
<strong>University</strong> <strong>of</strong> Illinois at <strong>Chicago</strong><br />
900 S Ashland Avenue<br />
MBRB 4168 MC 567<br />
<strong>Chicago</strong>, IL, 60607<br />
United States<br />
amario3@uic.edu<br />
Roger Markwald<br />
Department <strong>of</strong> Regenerative Medicine<br />
& Cell Biology<br />
Medical <strong>University</strong> <strong>of</strong> South Carolina<br />
173 Ashey Avenue<br />
BSB-601, PO Box 250508<br />
Charleston, SC, 29403<br />
United States<br />
fabunane@musc.edu<br />
Cindy Martin<br />
Department <strong>of</strong> Medicine, Cardiology<br />
Division<br />
<strong>University</strong> <strong>of</strong> Minnesota<br />
420 Delaware St S.E.<br />
MMC 508<br />
Minneapolis, MN, 55455<br />
United States<br />
cmmartin@umn.edu<br />
James Martin<br />
Department <strong>of</strong> Molecular Physiology<br />
Baylor College <strong>of</strong> Medicine<br />
One Baylor Plaza<br />
Room 414B<br />
Houston , Texas, 77030<br />
United States<br />
jfmartin@bcm.edu<br />
Silvia Martin-Puig<br />
Cardiovascular and Developmental<br />
Repair Department<br />
Fundacion CNIC<br />
Calle Melchor Fernandez Almagro, 3<br />
Madrid, Madrid, 28029<br />
Spain<br />
silvia.martin@cnic.es<br />
Cheryl Maslen<br />
Department <strong>of</strong> Cardiovascular Medicine<br />
and Molecular & Medical Genetics<br />
Oregon Health & Science <strong>University</strong><br />
CH14M<br />
3303 SW Bond St<br />
Portland, OR, 97239<br />
United States<br />
maslenc@ohsu.edu<br />
Andrew McKay<br />
Department <strong>of</strong> Biology<br />
Stanford <strong>University</strong><br />
Department <strong>of</strong> Biology<br />
371 Serra Mall<br />
Stanford, CA, 94305-5020<br />
United States<br />
andrew.s.mckay@gmail.com<br />
Saoirse McSharry<br />
Department <strong>of</strong> Medicine/ Cardiology<br />
The <strong>University</strong> <strong>of</strong> <strong>Chicago</strong><br />
5841 S. Maryland Ave<br />
<strong>Chicago</strong>, IL, 60637<br />
United States<br />
saoirse@uchicago.edu<br />
Sigolene Meilhac<br />
Department <strong>of</strong> Developmental Biology<br />
Institut Pasteur<br />
25 rue du Dr Roux<br />
Paris, , 75015<br />
France<br />
sigolene.meilhac@pasteur.fr<br />
Catherine Mercer<br />
Department <strong>of</strong> Human Genetics<br />
<strong>University</strong> <strong>of</strong> Southampton<br />
Duthie Building<br />
Southampton General Hospital<br />
Tremona Road, Hampshire, SO16 6YD<br />
United Kingdom<br />
catherinemercer@yahoo.com<br />
Sarah Mercer<br />
Department <strong>of</strong> Pediatrics<br />
Northwestern <strong>University</strong> and Children's<br />
Memorial Research Center<br />
2430 N. Halsted St<br />
<strong>Chicago</strong>, IL, 60614<br />
United States<br />
sarahmercer2013@u.northwestern.edu<br />
Takashi Mikawa<br />
Cardiovascular Research Institute<br />
<strong>University</strong> <strong>of</strong> California San Francisco<br />
555 Mission Bay Blvd South, MC3120<br />
San Francisco, CA, CA 94158-9001<br />
United States<br />
takashi.mikawa@ucsf.edu<br />
Paul Miller<br />
Department <strong>of</strong> Medicine/ Division <strong>of</strong><br />
Cardiovascular Medicine<br />
Vanderbilt Medical Center<br />
2220 Pierce Avenue<br />
PRB 346<br />
Nashville, TN, 37221<br />
United States<br />
paul.m.miller@vanderbilt.edu<br />
Lucile Miquerol<br />
Department <strong>of</strong> IBMDL CNRS UMR7288<br />
Aix-Marseille <strong>University</strong><br />
campus de Luminy- case 907<br />
Marseille, , 13288 cedex 9<br />
France<br />
lucile.miquerol@ibdml.univmed.fr<br />
189<br />
Chaitali Misra<br />
Center for Cardiovascular and<br />
Pulmonary Research<br />
Research Institute at Nationwide<br />
Children's Hospital<br />
700 Children's Drive<br />
Columbus, Ohio, 43205<br />
United States<br />
Chaitali.Misra@nationwidechildrens.org<br />
Ravi Misra<br />
Department <strong>of</strong> Biochemistry<br />
Medical College <strong>of</strong> Wisconsin<br />
8701 Watertown Plank Road<br />
Milwaukee, Wisconsin, 53226<br />
United States<br />
rmisra@mcw.edu<br />
Suniti Misra<br />
Department <strong>of</strong> Regenerative Medicine<br />
& Cell Biology<br />
Medical <strong>University</strong> <strong>of</strong> South Carolina<br />
BSB-601, PO Box 250508<br />
173 Ashley Avenue<br />
Charleston, SC, 29403<br />
United States<br />
misra@musc.edu<br />
Seema Mital<br />
Department <strong>of</strong> Pediatrics<br />
Hospital for Sick Children<br />
555 <strong>University</strong> Avenue<br />
Toronto, Ontario, M5G 1X8<br />
Canada<br />
seema.mital@sickkids.ca<br />
Grant Miura<br />
Division <strong>of</strong> Biological Sciences<br />
<strong>University</strong> <strong>of</strong> California, San Diego<br />
9500 Gilman Drive #0347<br />
La Jolla, CA, 92093-0347<br />
United States<br />
gmiura@ucsd.edu<br />
Corey Mjaatvedt<br />
Department <strong>of</strong> Regenerative Medicine<br />
and Cell Biology<br />
Medical <strong>University</strong> <strong>of</strong> South Carolina<br />
173 Ashley Ave - BSB 642<br />
MSC 508<br />
Charleston, SC, 29425<br />
United States<br />
mjaatvedt@musc.edu<br />
Hoda Moazzen<br />
Department <strong>of</strong> Physiology and<br />
Pharmacology<br />
<strong>University</strong> <strong>of</strong> Western Ontario<br />
Medical Sciences Building Rm216<br />
London, Ontario, N6A5C1<br />
Canada<br />
hmoazzen@uwo.ca
Mariya Mollova<br />
Department <strong>of</strong> Cardiology<br />
Children's Hospital Boston/ Harvard<br />
Medical School<br />
300 Longwood Avenue<br />
Boston, MA, 02115<br />
United States<br />
mmollova@enders.tch.harvard.edu<br />
Mathilda Mommersteeg<br />
Department <strong>of</strong> Cell and Developmental<br />
Biology<br />
<strong>University</strong> College London<br />
Gower street<br />
London, London, WC1E 6BT<br />
United Kingdom<br />
tilly75@hotmail.com<br />
Ricardo Moreno<br />
Department <strong>of</strong> Regenerative Medicine<br />
and Cardiovascular Biology Center<br />
Medical <strong>University</strong> <strong>of</strong> South Carolina.<br />
173 Ashley Avenue<br />
Children's Research Institute 604F<br />
Charleston , SC, 29425<br />
United States<br />
morenor@musc.edu<br />
Yuka Morikawa<br />
Department <strong>of</strong> Cardiomyocyte Renewal<br />
Texas Heart Institute<br />
6770 Bernter Ave<br />
MC 2-255<br />
Houston, TX, 77030<br />
ymorikawa@texasheart.org<br />
Yuika Morita<br />
Department <strong>of</strong> Cardiovascular<br />
Regeneration, IMCB<br />
the <strong>University</strong> <strong>of</strong> Tokyo<br />
1-1-1 Yayoi<br />
Bunkyo-ku, Tokyo, 113-0032<br />
Japan<br />
firecracker0109@yahoo.co.jp<br />
Bernice Morrow<br />
Department <strong>of</strong> Genetics<br />
Albert Einstein College <strong>of</strong> Medicine<br />
1301 Morris Park Ave<br />
Bronx, NY, 10461<br />
United States<br />
bernice.morrow@einstein.yu.edu<br />
Ivan Moskowitz<br />
Department <strong>of</strong> Pediatrics and Pathology<br />
The <strong>University</strong> <strong>of</strong> <strong>Chicago</strong><br />
900 East 57th Street, KCBD Rm 5118<br />
<strong>Chicago</strong>, IL, 60637<br />
United States<br />
imoskowitz@uchicago.edu<br />
Yosuke Mukoyama<br />
Laboratory <strong>of</strong> Stem Cell and Neuro-<br />
Vascular Biology, Genetics and<br />
Developmet Biology Center<br />
National Heart, Lung, and Blood<br />
Institute<br />
10 Center Dr. Room 6C103A<br />
Bethesda, Maryland, 20892<br />
United States<br />
mukoyamay@mail.nih.gov<br />
Charu Munjal<br />
heart institute<br />
cincinnati hospital<br />
3333 burnet ave<br />
mlc 7020<br />
cincinnati, OH<br />
United States<br />
charu.munjal@cchmc.org<br />
Nikhil Munshi<br />
Department <strong>of</strong> Internal Medicine -<br />
Cardiology<br />
UT Southwestern Medical Center<br />
5323 Harry Hines Blvd<br />
Dallas, TX, 75390<br />
United States<br />
nikhil.munshi@utsouthwestern.edu<br />
Agnes Nagy-Mehesz<br />
Department <strong>of</strong> Regenerative Medicine<br />
and Cell Biology<br />
Medical <strong>University</strong> <strong>of</strong> South Carolina<br />
173 Ashley Avenue CRI605<br />
Mailstop 250508<br />
Charleston, SC, 29425<br />
United States<br />
nagymehe@musc.edu<br />
Ryo Nakamura<br />
Division <strong>of</strong> Cardiovascular<br />
Regeneration, Institute <strong>of</strong> Molecular<br />
and Cellular Biosciences<br />
The <strong>University</strong> <strong>of</strong> Tokyo<br />
1-1-1<br />
Yayoi,Bunkyo-ku<br />
Tokyo, , 113-0032<br />
Japan<br />
ryon422@gmail.com<br />
Haruko Nakano<br />
Department <strong>of</strong> Molecular, Cell and<br />
Developmental Biology<br />
<strong>University</strong> <strong>of</strong> California, Los Angeles<br />
621 Charles E. Young Dr. South<br />
LSB Rm2204<br />
Los Angeles, CA, 90095<br />
United States<br />
hnakano@ucla.eud<br />
Zachary Neeb<br />
Department <strong>of</strong> Pediatrics<br />
Indiana <strong>University</strong> Purdue <strong>University</strong><br />
Indianapolis<br />
Indianapolis, IN, 46202<br />
United States<br />
zneeb@iupui.edu<br />
190<br />
Daryl Nelson<br />
Department <strong>of</strong> Cell and Regenerative<br />
Biology<br />
<strong>University</strong> <strong>of</strong> Wisconsin-Madison<br />
1300 <strong>University</strong> Ave<br />
Madison, Wisconsin, 53706<br />
United States<br />
donelson@wisc.edu<br />
Karen Niederreither<br />
Department <strong>of</strong> Nutritional Sciences,<br />
The <strong>University</strong> <strong>of</strong> Texas Austin<br />
Dell Pediatric Research Institute<br />
1400 Barbara Jordan Blvd.<br />
Austin, Texas, 78723-3092<br />
United States<br />
karenn@austin.utexas.edu<br />
Vishal Nigam<br />
Department <strong>of</strong> Pediatrics (Cardiology)<br />
<strong>University</strong> <strong>of</strong> California San Diego<br />
Box 0731<br />
9500 Gilman Drive<br />
San Diego, CA, 92093<br />
United States<br />
vnigam@ucsd.edu<br />
Julia Norden<br />
Institute for Molecular Biology<br />
Hannover Medical School<br />
Carl-Neuberg-Strasse 1<br />
Hannover, , 30625<br />
Germany<br />
norden.julia@mh-hannover.de<br />
Russell Norris<br />
Department <strong>of</strong> Regenerative Medicine<br />
& Cell Biology<br />
Medical <strong>University</strong> <strong>of</strong> South Carolina<br />
171 Ashley Avenue<br />
BSB-601<br />
Charleston, South Carolina, 29425<br />
United States<br />
norrisra@musc.edu<br />
Harold Olivey<br />
Department <strong>of</strong> Biology<br />
Indiana <strong>University</strong> Northwest<br />
3400 Broadway<br />
Department <strong>of</strong> Biology, MH338<br />
Gary, IN, 46408<br />
United States<br />
holivey@iun.edu<br />
Appledene Osbourne<br />
Department <strong>of</strong> Medicine<br />
<strong>University</strong> <strong>of</strong> <strong>Chicago</strong><br />
5841 S. Maryland Ave.<br />
MC 6088, G-603<br />
<strong>Chicago</strong>, IL, 60637<br />
United States<br />
appledene@uchicago.edu
Noelle Paffett-Lugassy<br />
Department <strong>of</strong> Cardiovascular<br />
Research Center<br />
Massachusetts General Hospital<br />
149 13th Street, Fourth Floor<br />
CNY 149, CVRC<br />
Charlestown, MA, 02139<br />
United States<br />
npaffett-lugassy@partners.org<br />
Sharina Palencia-Desai<br />
Department <strong>of</strong> Developmental Biology<br />
Cincinnati Children's Hospital Medical<br />
Center<br />
3333 Burnet Ave<br />
Cincinnati, OH, 45229<br />
United States<br />
sharina.desai@cchmc.org<br />
Daniel Palmer<br />
pardhananip@winthrop.edu<br />
Pooja Pardhanani<br />
Department <strong>of</strong> Biology<br />
Winthrop <strong>University</strong><br />
207 Dalton Hall<br />
Rock Hill, SC, 29733<br />
United States<br />
pardhananip@winthrop.edu<br />
Chinmoy Patra<br />
Department <strong>of</strong> Cardiac Development<br />
and Regeneration<br />
Max-Planck-Institute for Heart and Lung<br />
Research<br />
Parkstrasse-1<br />
Bad Nauheim, Hessen, 61231<br />
Germany<br />
chinmoy.patra@mpi-bn.mpg.de<br />
Louisa Petchey<br />
Department <strong>of</strong> Molecular Medicine Unit<br />
UCL Institute <strong>of</strong> Child Health<br />
30 Guilford Street<br />
London, , WC1N 1EH<br />
United Kingdom<br />
louisa.petchey.09@ucl.ac.uk<br />
Lindsy Peterson<br />
Department <strong>of</strong> Biomedical Engineering<br />
Case Western Reserve <strong>University</strong><br />
Wearn Lab Univ Hospital<br />
2053 Abington Rd<br />
Cleveland, OH, 44106<br />
United States<br />
lmp42@case.edu<br />
Elise Pfaltzgraff<br />
Department <strong>of</strong> Cell and Developmental<br />
Biology<br />
Vanderbilt <strong>University</strong><br />
2213 Garland Ave<br />
MRBIV 9415<br />
Nashville, TN, 37232<br />
United States<br />
elise.r.pfaltzgraff@vanderbilt.edu<br />
Jess Plavicki<br />
Department <strong>of</strong> School <strong>of</strong> Pharmacy<br />
<strong>University</strong> <strong>of</strong> Wisconsin at Madison<br />
777 Highland Avenue<br />
Madison, WI, 53705<br />
United States<br />
jsberndt2@wisc.edu<br />
Renita Polk<br />
Department <strong>of</strong> Physiology, Institute <strong>of</strong><br />
Genetic Medicine<br />
Johns Hopkins <strong>University</strong> School <strong>of</strong><br />
Medicine<br />
Biophysics 203<br />
725 N. Wolfe Street<br />
Baltimore, Maryland, 21205<br />
United States<br />
rpolk2@jhmi.edu<br />
George Porter<br />
Department <strong>of</strong> Pediatrics<br />
<strong>University</strong> <strong>of</strong> Rochester School <strong>of</strong><br />
Medicine<br />
Pediatric Cardiology<br />
601 Elmwood Ave., Box 631<br />
Rochester, NY, 14642<br />
United States<br />
george_porter@urmc.rochester.edu<br />
Kenneth Poss<br />
Department <strong>of</strong> Cell Biology<br />
Duke <strong>University</strong> Medical Center<br />
353 Nanaline Duke Building, Research<br />
Drive, Box 3709<br />
Durham, North Carolina, 27710<br />
United States<br />
kenneth.poss@duke.edu<br />
Alex Postma<br />
Department <strong>of</strong> Anatomy, Embryology &<br />
Phsyiology<br />
Academic Medical Center<br />
meibergdreef 15<br />
amsterdam, nh, 1105az<br />
Netherlands<br />
a.v.postma@amc.uva.nl<br />
Vladimir Potaman<br />
Department <strong>of</strong> Stem Cell Engineering<br />
Texas Heart Institute<br />
6770 Bertner Ave<br />
Houston, Texas, 77030<br />
United States<br />
vpotaman@texasheart.org<br />
Adam Prickett<br />
Department <strong>of</strong> Medical and Molecular<br />
Genetics<br />
King's College London<br />
8th Floor Tower Wing<br />
Guy's Hospital<br />
London, UK, SE1 9RT<br />
United Kingdom<br />
adam.prickett@kcl.ac.uk<br />
191<br />
William Pu<br />
Department <strong>of</strong> Cardiology<br />
Children's Hospital Boston<br />
300 Longwood Ave<br />
Boston, MA, 02115<br />
United States<br />
wpu@pulab.org<br />
Michel Puceat<br />
Department <strong>of</strong> Cardiology<br />
INSERM<br />
4 rue Pierre Fontaine<br />
campus Genopole 1<br />
EVRY, ile de France, 91058<br />
France<br />
michel.puceat@inserm.fr<br />
Silvia Racedo<br />
Department <strong>of</strong> Genetics<br />
Albert Einstein College <strong>of</strong> Medicine<br />
1301 Morris Park Ave R408<br />
Bronx, New York, 10461<br />
United States<br />
silvia.racedo@einstein.yu.edu<br />
Sudha Rajderkar<br />
Department <strong>of</strong> Biologic Sciences,<br />
School <strong>of</strong> Dentistry<br />
The <strong>University</strong> <strong>of</strong> Michigan<br />
1011 N <strong>University</strong> #2310 A<br />
Ann Arbor, MI, 48109<br />
United States<br />
rsudha@umich.edu<br />
M. Sameer Rana<br />
Department <strong>of</strong> Heart Failure Research<br />
Center<br />
Academic Medical Center<br />
Meibergdreef 15<br />
Amsterdam, North Holland, 1105AZ<br />
Netherlands<br />
m.s.rana@amc.uva.nl<br />
Kristen Red-Horse<br />
Department <strong>of</strong> Biology<br />
Stanford <strong>University</strong><br />
Department <strong>of</strong> Biology<br />
371 Serra Mall<br />
Stanford, CA, 94305-5020<br />
United States<br />
kredhors@stanford.edu<br />
Roger Reeves<br />
Department <strong>of</strong> Dept. <strong>of</strong> Physiology and<br />
Institute for Genetic Medicine<br />
Johsn Hopkins <strong>University</strong> School <strong>of</strong><br />
Medicine<br />
725 N Wolfe ST.<br />
Biophysics 201<br />
JHU SOM, Maryland, Baltimore/ 2120<br />
United States<br />
rreeves@jhmi.edu
Stacey Rentschler<br />
Department <strong>of</strong> Medicine<br />
<strong>University</strong> <strong>of</strong> Pennsylvania<br />
1143 BRB II/III<br />
421 Curie Blvd<br />
Philadelphia, PA, 19104<br />
United States<br />
stacey.rentschler@uphs.upenn.edu<br />
Silke Rickert-Sperling<br />
Experimental and Clinical Research<br />
Center - Cardiovascular Genetics<br />
Charité Medical Faculty and Max<br />
Delbrück Center for Molecular Medicine<br />
Lindenberger Weg 80<br />
Berlin, Germany, 13125<br />
silke.sperling@charite.de<br />
Kevin Rigby<br />
Department <strong>of</strong> Molecular Medicine<br />
<strong>University</strong> <strong>of</strong> Utah School <strong>of</strong> Medicine<br />
15 N 2030 E Bldg. 533 Rm 4270<br />
Salt Lake City, UT, 84112<br />
United States<br />
rigby@u2m2.utah.edu<br />
Paul Riley<br />
Department <strong>of</strong> Physiology, Anatomy<br />
and Genetics<br />
<strong>University</strong> <strong>of</strong> Oxford<br />
Sherrington Building<br />
South Parks Road<br />
Oxford, United Kingdom<br />
OX1 3PT<br />
paul.riley@dpag.ox.ac.uk<br />
Andrew Robson<br />
Department <strong>of</strong> Pediatrics<br />
Yale <strong>University</strong><br />
333 Cedar Street<br />
FMP425<br />
New Haven, CT, 06520<br />
United States<br />
andrew.robson@yale.edu<br />
Brenda Rongish<br />
Department <strong>of</strong> Anatomy and Cell<br />
Biology<br />
<strong>University</strong> <strong>of</strong> Kansas Medical Center<br />
MS 3038<br />
3901 Rainbow Blvd.<br />
Kansas City, KS, 66160<br />
United States<br />
brongish@kumc.edu<br />
Jonathan Rosen<br />
Department <strong>of</strong> Cardiology<br />
Children's Hospital Boston<br />
300 Longwood Avenue<br />
Boston, MA, 02115<br />
United States<br />
rosen.jonathan@gmail.com<br />
Carsten Rudat<br />
Institute for Molecularbiology<br />
Hannover Medical School<br />
Carl-Neuberg-Str. 1<br />
Hannover, Lower Saxony, 30625<br />
Germany<br />
rudat.carsten@mh-hannover.de<br />
Raymond Runyan<br />
Department <strong>of</strong> Cellular and molecular<br />
Medicine<br />
<strong>University</strong> <strong>of</strong> Arizona<br />
1501 N. Campbell Ave., LSN 456<br />
Tucson, Arizona, 85724-5044<br />
United States<br />
rrunyan@email.arizona.edu<br />
Ariel Rydeen<br />
Department <strong>of</strong> Molecular<br />
Cardiovascular Biology<br />
Cincinnati Children's Hospital<br />
3333 Burnet Ave<br />
Cincinnati, Ohio, 45229<br />
United States<br />
rydeenal@mail.uc.edu<br />
Rudyard Sadleir<br />
Department <strong>of</strong> Pediatrics<br />
Northwestern <strong>University</strong> Feinberg<br />
School <strong>of</strong> Medicine<br />
Children's Memorial Research Center<br />
2430 N. Halsted<br />
<strong>Chicago</strong>, IL, 60614<br />
United States<br />
rudyardw@gmail.com<br />
Yukio Saijoh<br />
Department <strong>of</strong> Neurobiology and<br />
Anatomy<br />
<strong>University</strong> <strong>of</strong> Utah<br />
20 North 1900 East<br />
401 MREB<br />
Salt Lake City, Utah, 84112-3401<br />
United States<br />
y.saijoh@utah.edu<br />
Juan Jose Sanz-Ezquerro<br />
Cardiovascular Development and<br />
Regeneration<br />
CNIC (Centro Nacional de<br />
Investigaciones Cardiovasculares)<br />
Melchor Frenandez Almagro, 3<br />
Madrid, Spain, 28029<br />
jjsanz@cnic.es<br />
Rachel Sarig<br />
Department <strong>of</strong> Biological Regulation<br />
Weizmann Institute <strong>of</strong> Science<br />
Herzel St.<br />
Rehovot, Israel, 76100<br />
rachel.sarig@weizmann.ac.il<br />
192<br />
Kimberly Sauls<br />
Department Of Regenerative Medicine<br />
& Cell Biology<br />
Medical <strong>University</strong> Of South Carolina<br />
171 Ashley Avenue<br />
Bsb-601<br />
Charleston, South Carolina, 29425<br />
United States<br />
saulsk@musc.edu<br />
Charlene Schramm<br />
Division <strong>of</strong> Cardiovascular Sciences<br />
National Heart Lung and Blood institute<br />
6701 Rockledge Drive<br />
Bethesda, Maryland, 20892<br />
United States<br />
schramm@mail.nih.gov<br />
Claire Schulkey<br />
Department <strong>of</strong> Computational and<br />
Systems Biology<br />
Washington <strong>University</strong> School <strong>of</strong><br />
Medicine<br />
660 S. Euclid<br />
Saint Louis, mo, 63112<br />
United States<br />
ceschulk@wustl.edu<br />
Marcus-Oliver Schupp<br />
Department <strong>of</strong> Pediatrics /<br />
Developmental Biology<br />
Medical College <strong>of</strong> Wisconsin<br />
8701 W Watertown Plank Rd<br />
Milwaukee, WI, 53226<br />
United States<br />
mschupp@mcw.edu<br />
Ian Scott<br />
Department <strong>of</strong> Developmental and<br />
Stem Cell Biology<br />
The Hospital for Sick Children<br />
555 <strong>University</strong> Avenue<br />
Toronto, Ontario, M5G1X8<br />
Canada<br />
ian.scott@sickkids.ca<br />
Jonathan Seidman<br />
Department <strong>of</strong> Genetics<br />
Harvard Medical School<br />
Dept Genetics, NRB-256<br />
77 Avenue Louis Pasteur<br />
Boston, MA, 02115<br />
United States<br />
seidman@genetics.med.harvard.edu<br />
Parisha Shah<br />
Department <strong>of</strong> Pediatric Cardiology<br />
Stanford <strong>University</strong> School <strong>of</strong> Medicine<br />
750 Welch Road #325<br />
Palo Alto, CA, 94304<br />
United States<br />
pbshah@stanford.edu
Hua Shen<br />
Eli and Edythe Broad Center for<br />
Regenerative Medicine and Stem Cell<br />
<strong>University</strong> <strong>of</strong> Southern California<br />
1425 San Pablo St<br />
Los Angeles, California, 90033<br />
United States<br />
huashen@usc.edu<br />
Kyoo Shim<br />
Feinberg Cardiovascular Research<br />
Institute<br />
Northwestern <strong>University</strong><br />
303 E. <strong>Chicago</strong> Ave.<br />
Tarry 14-722/724<br />
<strong>Chicago</strong>, IL, 60611<br />
United States<br />
ks-shim@northwestern.edu<br />
Hirohito Shimizu<br />
Department <strong>of</strong> Molecular, Cell and<br />
Developmental Biology<br />
UCLA<br />
615 Charles E. Young Drive South<br />
Los Angeles, CA, 90095<br />
United States<br />
hshimizu@ucla.edu<br />
Isao Shiraishi<br />
Department <strong>of</strong> Pediatric Cardiology<br />
National Cerebral and Cardiovascular<br />
Center<br />
5-7-1, Fujishirodai<br />
, Osaka, Siuta<br />
Japan<br />
isao@hsp.ncvc.go.jp<br />
Weinian Shou<br />
Department <strong>of</strong> Pediatrics<br />
Indiana <strong>University</strong> School <strong>of</strong> Medicine,<br />
Wells Center for Pediatric Research<br />
1044 W Walnut, R4-302<br />
Indianapolis, IN, 46202<br />
United States<br />
wshou@iupui.edu<br />
Rickert-Sperling Silke<br />
Department <strong>of</strong> Cardiovascular Genetics<br />
Experimental and Clinical Research<br />
Center <strong>of</strong> Charité Medical Faculty and<br />
Max Delbrück Center for Molecular<br />
Medicine<br />
Lindenberger Weg 80<br />
Berlin, Germany, 13125<br />
silke.sperling@charite.de<br />
Brynn Simek<br />
Department <strong>of</strong> Biology (Institute <strong>of</strong><br />
Molecular Biology)<br />
<strong>University</strong> <strong>of</strong> Oregon<br />
1370 Franklin Blvd.<br />
Room 266 Klamath Hall<br />
Eugene, OR, 97403<br />
United States<br />
bsimek@uoregon.edu<br />
Hans-Georg Simon<br />
Department <strong>of</strong> Pediatrics<br />
Northwestern <strong>University</strong> Feinberg<br />
School <strong>of</strong> Medicine and Children's<br />
Memorial Research Center<br />
CMRC<br />
2430 N. Halsted Street<br />
<strong>Chicago</strong>, Illinois, 60614<br />
United States<br />
hgsimon@northwestern.edu<br />
Manvendra Singh<br />
Department <strong>of</strong> Cell and Developmental<br />
Biology<br />
<strong>University</strong> <strong>of</strong> Pennsylvania<br />
1139, Biomedical Rsch Bldg (BRB) II/III<br />
421 Curie Boulevard<br />
Philadelphia, PA, 19104<br />
United States<br />
masingh@mail.med.upenn.edu<br />
Tanvi Sinha<br />
Department <strong>of</strong> Cell, Developmental and<br />
Integrative Biology<br />
<strong>University</strong> <strong>of</strong> Alabama at Birmingham<br />
MCLM 305<br />
19 18 <strong>University</strong> Blvd.<br />
Birmingham, Alabama, 35294<br />
United States<br />
tsinha@uab.edu<br />
Scott Smemo<br />
Department <strong>of</strong> Human Genetics<br />
<strong>University</strong> <strong>of</strong> <strong>Chicago</strong><br />
920 E 58th ST<br />
CLSC 301<br />
<strong>Chicago</strong>, IL, 60637<br />
United States<br />
ssmemo@uchicago.edu<br />
Kelly Smith<br />
Institute for Molecular Bioscience<br />
The <strong>University</strong> <strong>of</strong> Queensland<br />
306 Carmody Road<br />
St Lucia<br />
Brisbane, Queensland, 4072<br />
Australia<br />
k.smith@imb.uq.edu.au<br />
Paige Snider<br />
Department <strong>of</strong> Pediatrics<br />
Indiana <strong>University</strong> Purdue <strong>University</strong><br />
Indianapolis<br />
1044 W. Walnut Street<br />
R4 402F<br />
Indianapolis, IN, 46202<br />
United States<br />
psnider@iupui.edu<br />
Stefanovic Sonia<br />
Department <strong>of</strong> Anatomy, Embryology &<br />
Physiology<br />
Academic Medical Center, <strong>University</strong> <strong>of</strong><br />
Amsterdam<br />
Meibergdreef 15<br />
Amsterdam , Netherlands, 1105 AZ<br />
s.stefanovic@amc.uva.nl<br />
193<br />
Daniela Spaeter<br />
Cardiovascular Research Center<br />
Massachusetts General Hospital<br />
185 Cambridge Street CPZN3208<br />
Boston, Massachusetts , 02114<br />
United States<br />
dspaeter@gmx.net<br />
Matthew Spindler<br />
Insitute <strong>of</strong> Cardiovascular Disease<br />
The J. David Gladstone Institutes<br />
1650 Owens Street<br />
San Francisco, CA, 94158<br />
United States<br />
mspindler@gladstone.ucsf.edu<br />
Kryn Stankunas<br />
Institute <strong>of</strong> Molecular Biology<br />
<strong>University</strong> <strong>of</strong> Oregon<br />
297 Klamath Hall<br />
1229 <strong>University</strong> <strong>of</strong> Oregon<br />
Eugene, Oregon, 97403<br />
United States<br />
kryn@uoregon.edu<br />
Sonia Stefanovic<br />
Department <strong>of</strong> Anatomy, Embryology &<br />
Physiology<br />
Academic Medical Center, <strong>University</strong> <strong>of</strong><br />
Amsterdam<br />
Meibergdreef 15 (L2-109)<br />
Amsterdam , Netherlands, 1105 AZ<br />
S.Stefanovic@amc.uva.nl<br />
Jason Stoller<br />
Department <strong>of</strong> Pediatrics/Neonatology<br />
Children's Hospital <strong>of</strong> Philadelphia<br />
3615 Civic Center Blvd, Suite 416F<br />
Philadelphia, PA, 19104<br />
United States<br />
stoller@email.chop.edu<br />
Henry Sucov<br />
Broad CIRM Center<br />
<strong>University</strong> <strong>of</strong> Southern California<br />
1425 San Pablo St., BCC-511,<br />
Mail Code 9080<br />
Los Angeles, CA, 90033<br />
United States<br />
sucov@usc.edu<br />
Yukiko Sugi<br />
Department <strong>of</strong> Regenerative Medicine<br />
and Cell Biology<br />
Medical <strong>University</strong> <strong>of</strong> South Carolina<br />
171 Ashley Avenue<br />
Charleston, SC, 29425<br />
United States<br />
sugiy@musc.edu<br />
Saulius Sumanas<br />
Division <strong>of</strong> Developmental Biology<br />
Cincinnati Children's Hospital Medical<br />
Center<br />
3333 Burnet Ave<br />
Cincinnati, OH, 45229<br />
United States<br />
saulius.sumanas@cchmc.org
Mardi Sutherland<br />
Department <strong>of</strong> Molecular<br />
Cardiovascular Biology<br />
Cincinnati Children's Hospital<br />
240 Albert Sabin Way<br />
MLC 7020<br />
Cincinnati, OH, 45229<br />
United States<br />
mardi.sutherland@cchmc.org<br />
Eric Svensson<br />
Department <strong>of</strong> Medicine<br />
The <strong>University</strong> <strong>of</strong> <strong>Chicago</strong><br />
AMB G-611, MC6088<br />
5841 S. Maryland Ave<br />
<strong>Chicago</strong>, IL, 60637<br />
United States<br />
esvensso@medicine.bsd.uchicago.edu<br />
Marc Sylva<br />
Academic Medical Center<br />
Anatomy Embryology & Physiology<br />
Meibergdreef 15<br />
Amsterdam, Netherlands, 1105AZ<br />
M.Sylva@AMC.nl<br />
Marta Szmacinski<br />
Department <strong>of</strong> Genetics and Molecular<br />
Biology<br />
UNC-Chapel Hill<br />
221 Fordham Hall<br />
Chapel Hill, NC, 27517<br />
United States<br />
mszmac86@gmail.com<br />
Zhiping Tan<br />
Department <strong>of</strong> Cardiothoracic Surgery<br />
Clinical Center for Gene Diagnosis and<br />
Therapy <strong>of</strong> State Key Laboratory <strong>of</strong><br />
Medical Genetics, The Second Xiangya<br />
Hospital,<br />
139# Renmin Road, Changsha, Hunan<br />
410011, China<br />
Changsha, Hunan,<br />
China<br />
tanzhiping@gmail.com<br />
Jiayi Tao<br />
Department <strong>of</strong> Pediatrics<br />
Case Western Reserve <strong>University</strong><br />
10900 Euclid Avenue<br />
Cleveland, OH, 44106<br />
United States<br />
jxt314@case.edu<br />
Kimara Targ<strong>of</strong>f<br />
Department <strong>of</strong> Pediatrics<br />
Columbia <strong>University</strong><br />
College <strong>of</strong> Physicians & Surgeons<br />
630 West 168th Street<br />
New York, NY, 10032<br />
United States<br />
kl284@columbia.edu<br />
Federico Tessadori<br />
Department <strong>of</strong> Cardiac Development<br />
and Genetics<br />
Hubrecht Institute<br />
Uppsalalaan, 8<br />
Utrecht, Netherlands, 3584 CT<br />
federico_tessadori@yahoo.com<br />
Penny Thomas<br />
Department <strong>of</strong> Biologic and Materials<br />
Sciences<br />
<strong>University</strong> <strong>of</strong> Michigan<br />
1011 N <strong>University</strong> Avenue<br />
School <strong>of</strong> Dentistry<br />
Ann Arbor, MI, 48109-1078<br />
United States<br />
ptho@umich.edu<br />
Rebecca Thomason<br />
Department <strong>of</strong> Cell and Developmental<br />
Biology<br />
Vanderbilt <strong>University</strong><br />
2220 Pierce Ave, 348 PRB<br />
Nashville, TN, 37232<br />
United States<br />
rebecca.t.thomason@vanderbilt.edu<br />
Ying Tian<br />
Department <strong>of</strong> Medicine<br />
<strong>University</strong> <strong>of</strong> Pennsylvania<br />
3400 Civic Center Boulevard<br />
Translational Research Center, 11th<br />
Flr.; Room 179<br />
Philadelphia, PA, 19104<br />
United States<br />
yitian@mail.med.upenn.edu<br />
Sachiko Tomita<br />
Department <strong>of</strong> Pediatric Cardiology &<br />
Medical Research Institute<br />
Tokyo Women's Medical <strong>University</strong><br />
8-1 Kawada-cho<br />
Shinjuku-ku<br />
Tokyo, , 162-8666<br />
Japan<br />
ptomita@hij.twmu.ac.jp<br />
Miguel Torres<br />
Cardiovascular Development and<br />
Repair Department<br />
Fundacion CNIC<br />
Calle Melchor Fernandez Almagro, 3<br />
Madrid, Madrid, 28029<br />
Spain<br />
mtorres@cnic.es<br />
Todd Townsend<br />
Department <strong>of</strong> Neurobiology and<br />
Anatomy - Molecular Medicine Program<br />
<strong>University</strong> <strong>of</strong> Utah<br />
15 N 2130 E<br />
3260 EIHG<br />
Salt Lake City, Utah, 84112<br />
United States<br />
ttownsend@genetics.utah.edu<br />
194<br />
Harma Turbendian<br />
Department <strong>of</strong> Surgery<br />
Weill Cornell Medical College<br />
1300 York Avenue<br />
New York, NY, 10065<br />
United States<br />
hkt2002@med.cornell.edu<br />
Keiko Uchida<br />
Department <strong>of</strong> Pediatrics<br />
Keio <strong>University</strong> School <strong>of</strong> Medicine<br />
35 Shinanomachi<br />
Shinjuku-ku, Tokyo, 160-8582<br />
Japan<br />
uchida@muc.biglobe.ne.jp<br />
Ryan Udan<br />
Department <strong>of</strong> Molecular Physiology<br />
and Biophysics<br />
BCM (Baylor College <strong>of</strong> Medicine)<br />
1 Baylor Plaza, BCM MS 335<br />
Houston, TX,<br />
United States<br />
rudan@bcm.edu<br />
Hideki Uosaki<br />
Division <strong>of</strong> Cardiology<br />
Johns Hopkins <strong>University</strong> School <strong>of</strong><br />
Medicine<br />
720 Rutland Ave<br />
Ross 954<br />
Baltimore, MD, 21205<br />
United States<br />
huosaki1@jhmi.edu<br />
Maurice van den H<strong>of</strong>f<br />
Department <strong>of</strong> Anatomy, Embryology &<br />
Physiology<br />
Academic Medical Center - <strong>University</strong><br />
<strong>of</strong> Amsterdam<br />
Meibergdreef 15<br />
Amsterdam, Netherlands, 1105AZ<br />
m.j.vandenh<strong>of</strong>f@amc.uva.nl<br />
Sunil Verma<br />
Department <strong>of</strong> Biochemistry and<br />
Molecular Biology<br />
<strong>University</strong> <strong>of</strong> Texas Medical Branch<br />
Galveston<br />
301 <strong>University</strong> Blvd.<br />
Bld # 17, Room # 4.304<br />
Galveston, Texas, 77555<br />
United States<br />
skverma@utmb.edu<br />
Richard Visconti<br />
Department <strong>of</strong> Regenerative Medicine<br />
and Cell Biology<br />
Medical <strong>University</strong> <strong>of</strong> South Carolina<br />
173 Ashley Avenue - CRI607<br />
Mailstop 250508<br />
Charleston, SC, 29425<br />
United States<br />
visconrp@musc.edu
Georg Vogler<br />
Department <strong>of</strong> Neuroscience, Aging<br />
and Stem Cell Research Center<br />
Sanford-Burnham Medical Research<br />
Institute<br />
10901 N. Torrey Pines Rd<br />
La Jolla, CA, 92037<br />
United States<br />
gvogler@sanfordburnham.org<br />
Da-Zhi Wang<br />
Department <strong>of</strong> Cardiology<br />
Children's Hospital Boston, Harvard<br />
Medical School<br />
320 Longwood Avenue<br />
Boston, MA, 02115<br />
United States<br />
dwang@enders.tch.harvard.edu<br />
Jianbo Wang<br />
Department <strong>of</strong> Cell, Developmental and<br />
Integrative Biology<br />
Univ. <strong>of</strong> Alabama at Birmingham<br />
1918 <strong>University</strong> Blvd.<br />
MCLM 392<br />
Birmingham, AL, 35294<br />
United States<br />
j18wang@uab.edu<br />
Qun-Tian Wang<br />
Department <strong>of</strong> Biological Sciences<br />
<strong>University</strong> <strong>of</strong> Illinois at <strong>Chicago</strong><br />
900 S Ashland Avenue<br />
MBRB 4168 MC 567<br />
<strong>Chicago</strong>, IL, 60607<br />
United States<br />
qtwang@uic.edu<br />
Xia Wang<br />
Center for Translational Medicine<br />
Jefferson Medical College<br />
1025 walnut street<br />
Suit 301<br />
Philadelphia, PA, 19107<br />
United States<br />
xia.wang@jefferson.edu<br />
Maqsood Wani<br />
Center for Scientific Review<br />
National Institutes <strong>of</strong> Health<br />
4136 RKL II<br />
6701 Rockledge Dr<br />
Bethesda, MD, 20892<br />
United States<br />
wanimaqs@csr.nih.gov<br />
Stephanie Ware<br />
Divisions <strong>of</strong> Cardiology and Genetics<br />
Cincinnati Children's Hospital Medical<br />
Center<br />
3333 Burnet Avenue, MLC 7020<br />
Cincinnati, OH, 45229<br />
United States<br />
Stephanie.Ware@cchmc.org<br />
Michiko Watanabe<br />
Department <strong>of</strong> Pediatrics<br />
CWRU SOM--RB&C Hospital<br />
11100 Euclid Avenue<br />
Cleveland, OH, 44106<br />
United States<br />
mxw13@case.edu<br />
Joshua Waxman<br />
Molecular Cardiovascular Biology<br />
Division an Heart Institute<br />
Cincinnati Children's Hospital Medical<br />
Center<br />
240 Albert Sabin Way<br />
MLC7020, S4.234<br />
Cincinnati, OH, 45229<br />
United States<br />
joshua.waxman@cchmc.org<br />
Dan Weeks<br />
Department <strong>of</strong> Biochemistry<br />
<strong>University</strong> <strong>of</strong> Iowa<br />
Iowa City, Iowa, 52242<br />
United States<br />
daniel-weeks@uiowa.edu<br />
Kuo-Chan Weng<br />
Department <strong>of</strong> Stem Cell Engineering<br />
Texas Heart Institute<br />
6770 Bertner Ave<br />
MC2-255<br />
Houston, TX, 77030<br />
United States<br />
kweng@ibt.tamhsc.edu<br />
Andy Wessels<br />
Department <strong>of</strong> Regenerative Medicine<br />
and Cell Biology<br />
Medical <strong>University</strong> <strong>of</strong> South Carolina<br />
173 Ashley Avenue<br />
Charleston, South Carolina, 29425<br />
United States<br />
wesselsa@musc.edu<br />
Nichelle Winters<br />
Department <strong>of</strong> Cell and Developmental<br />
Biology<br />
Vanderbilt <strong>University</strong><br />
2220 Pierce Ave<br />
346 PRB<br />
Nashville, TN, 37232<br />
United States<br />
niki.winters@vanderbilt.edu<br />
Cindy Wong<br />
Scientific Business Development<br />
Abcam<br />
1 Kendall Square<br />
Bldg. 200, Suite B2304<br />
Cambridge, MA, 02139<br />
United States<br />
cindy.wong@abcam.com<br />
195<br />
Mingfu Wu<br />
Cardiovascular Science Center<br />
Albany Medical College<br />
ME616 MC8<br />
43 New Scotland Ave.<br />
Albany Medical College, Albany , NY,<br />
12208<br />
United States<br />
wum@mail.amc.edu<br />
Sean Wu<br />
Cardiovascular Research Center<br />
Massachusetts General Hospital<br />
Room 3224 Simches Building<br />
185 Cambridge Street<br />
Boston, MA, 02114<br />
United States<br />
smwu@partners.org<br />
Xiushan Wu<br />
The Center for Heart Development<br />
Hunan Normal <strong>University</strong><br />
Lushan Lu 36<br />
Changsha, Hunan, 410081<br />
China<br />
xiushanwu2003@yahoo.com.cn<br />
Eugene Wyatt<br />
Department <strong>of</strong> Medicine<br />
<strong>University</strong> <strong>of</strong> <strong>Chicago</strong><br />
AMB G-608<br />
5841 S Maryland Ave<br />
<strong>Chicago</strong>, IL, 60637<br />
United States<br />
ewyatt2@uchicago.edu<br />
Joshua Wythe<br />
Gladstone Institute <strong>of</strong> Cardiovascular<br />
Disease<br />
Gladstone/UCSF<br />
1650 Owens Street<br />
San Francisco, CA, 94158<br />
United States<br />
jwythe@gladstone.ucsf.edu<br />
Qi Xiao<br />
Model Animal Research Center<br />
Nanjing <strong>University</strong><br />
12 Xuefu Road, Pukou, Nanjing, China<br />
210061<br />
Nanjing, Jiangsu, 210061<br />
mac860622@gmail.com<br />
Linglin Xie<br />
Department <strong>of</strong> Biochemistry &<br />
Molecular Biology<br />
<strong>University</strong> <strong>of</strong> North Dakota<br />
501 N Columbia Road Stop 9037<br />
Grand Forks , ND, 58202-9037<br />
United States<br />
linglin.xie@med.und.edu
Hisato Yagi<br />
Department <strong>of</strong> Developmental Biology<br />
<strong>University</strong> <strong>of</strong> Pittsburgh<br />
530 45th street<br />
Pittsburgh, PA, 15201<br />
United States<br />
hisato@pitt.edu<br />
Hiroyuki Yamagishi<br />
Department <strong>of</strong> Pediatrics<br />
Keio <strong>University</strong> School <strong>of</strong> Medicine<br />
35 Shinanomachi, Shinjyuku-ku<br />
Tokyo, Japan, 160-8582<br />
hyamag@z6.keio.jp<br />
Yuki Yamaguchi<br />
Department <strong>of</strong> Biochemistry<br />
<strong>University</strong> <strong>of</strong> Southern California<br />
1425 San Pablo St<br />
Los Angeles, CA, 90033<br />
United States<br />
yukikoya@usc.edu<br />
PoAn Yang<br />
Cardiovascular Research Institute<br />
<strong>University</strong> <strong>of</strong> California San Francisco<br />
555 Mission Blvd/South<br />
San Francisco, CA, 94158<br />
United States<br />
byang80@mail.sfsu.edu<br />
Xinan Yang<br />
Department <strong>of</strong> Pediatrics<br />
the <strong>University</strong> <strong>of</strong> <strong>Chicago</strong><br />
900 East 57th Street, KCBD Rm 5121<br />
<strong>Chicago</strong>, IL, 60637<br />
United States<br />
xyang2@uchicago.edu<br />
Zhongzhou Yang<br />
Model Animal Research Center<br />
Nanjing <strong>University</strong><br />
12 Xuefu Road, Pukou<br />
Nanjing, Jiangsu, 210061<br />
China<br />
zhongzhouyang@nju.edu.cn<br />
Choon Hwai Yap<br />
Department <strong>of</strong> Developmental Biology,<br />
School <strong>of</strong> Medicine<br />
<strong>University</strong> <strong>of</strong> Pittsburgh<br />
530, 45th Street, 8119 Rangos<br />
Research Center<br />
Pittsburgh, PA, 15206<br />
United States<br />
cyap@pitt.edu<br />
Deborah Yelon<br />
Division <strong>of</strong> Biological Sciences<br />
<strong>University</strong> <strong>of</strong> California, San Diego<br />
9500 Gilman Drive, Mail Code #0347<br />
La Jolla, CA, 92093-0347<br />
United States<br />
dyelon@ucsd.edu<br />
H Joseph Yost<br />
Department <strong>of</strong> Molecular Medicine<br />
<strong>University</strong> <strong>of</strong> Utah<br />
15 N. 2030 East<br />
Suite 3160<br />
Salt Lake City, UT, 84112<br />
United States<br />
jyost@genetics.utah.edu<br />
Eugene Yu<br />
Department <strong>of</strong> Genetics Program<br />
Roswell Park Cancer Institute<br />
Center for Genetics and Pharmacology<br />
Rm 2320<br />
Elm and Carlton Streets<br />
Buffalo, NY, 14263<br />
United States<br />
yuejin.yu@roswellpark.org<br />
Baiyin Yuan<br />
Model Animal Research Center<br />
Nanjing <strong>University</strong><br />
Pukou,NanJing<br />
12 Xuefu Road<br />
NanJing, JiangSu, 210061<br />
China<br />
baiyinyuan@gmail.com<br />
Katherine Yutzey<br />
Department <strong>of</strong> Molecular<br />
Cardiovascular Biology<br />
Cincinnati Children's Medical Center<br />
ML7020<br />
240 Albert Sabin Way<br />
Cincinnati, OH, 45229<br />
United States<br />
katherine.yutzey@cchmc.org<br />
Stephane Zaffran<br />
Department <strong>of</strong> UMR_S910, Medical<br />
Genetics & Functional Genomics<br />
INSERM, Aix-Marseille <strong>University</strong><br />
School <strong>of</strong> Medicine<br />
27 Bd Jean Moulin<br />
Marseille, France, 13005<br />
stephane.zaffran@univ-amu.fr<br />
Kurt Zhang<br />
Department <strong>of</strong> Pathology<br />
<strong>University</strong> <strong>of</strong> North Dakota School <strong>of</strong><br />
Medicine<br />
501 N. Columbia Rd Stop 9037<br />
Grand Forks, ND, 58202-9037<br />
United States<br />
ke.zhang@med.und.edu<br />
Ruilin zhang<br />
Department <strong>of</strong> Medicine<br />
UCSD<br />
9500 Gilman Dr<br />
GPL 100A<br />
La Jolla, California, 92122<br />
United States<br />
r7zhang@ucsd.edu<br />
196<br />
Wenjun Zhang<br />
Department <strong>of</strong> Pediatrics<br />
Indiana <strong>University</strong> Purdue <strong>University</strong><br />
Indianapolis<br />
1044 W. Walnut Street<br />
R4 402F<br />
Indianapolis, IN, 46202<br />
United States<br />
wenzhang@iupui.edu<br />
Tao Zhong<br />
Department <strong>of</strong> Cardiovascular Medicine<br />
Vanderbilt <strong>University</strong><br />
358 PRB, 2220 Pierce Avenue<br />
Nashville, TN, 37203<br />
United States<br />
tao.p.zhong@vanderbilt.edu<br />
Maggie Zhu<br />
School <strong>of</strong> Medicine<br />
UCSD<br />
9500 Gilman Dr<br />
La Jolla, CA, 92092<br />
United States<br />
x3zhu@ucsd.edu<br />
Claudia Zierold<br />
Department <strong>of</strong> Medicine<br />
Lillehei Heart Institute <strong>University</strong> <strong>of</strong><br />
Minnesota<br />
312 Church St SE<br />
Minneapolis , MN, 55455<br />
United States<br />
ziero003@umn.edu
Notes<br />
197
198
199
200
See you at next year’s <strong>Weinstein</strong> Conference!<br />
2013 <strong>Weinstein</strong> Cardiovascular Development Conference<br />
May 16-18, 2013<br />
Tucson, Arizona
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